CA3056129A1 - Modulators of pcsk9 expression - Google Patents

Modulators of pcsk9 expression Download PDF

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CA3056129A1
CA3056129A1 CA3056129A CA3056129A CA3056129A1 CA 3056129 A1 CA3056129 A1 CA 3056129A1 CA 3056129 A CA3056129 A CA 3056129A CA 3056129 A CA3056129 A CA 3056129A CA 3056129 A1 CA3056129 A1 CA 3056129A1
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compound
certain embodiments
isis
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Eric E. Swayze
Susan M. Freier
Huynh-Hoa Bui
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Ionis Pharmaceuticals Inc
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Ionis Pharmaceuticals Inc
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Abstract

The present embodiments provide methods, compounds, and compositions useful for inhibiting PCSK9 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PCSK9.

Description

Sequence Listing The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 200615-WO-PCT-SequenceListing.txt created February 16, 2018, which is 431 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
Field The present embodiments provide methods, compounds, and compositions useful for inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, and in certain instances, reducing the amount of PCSK9 protein in a cell or animal, which can be useful for treating, preventing, or ameliorating a disease associated with PCSK9.
Background Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme with an important role in lipoprotein metabolism. Rare gain-of-function mutations in PCSK9 lead to a high LDL-C level and premature coronary heart disease, whereas loss-of-function variants lead to a low LDL-C
level and a reduced incidence of coronary heart disease (Zhao et al., Am. J. Hum. Genet. 2006,79: 514-523;
Horton et al., J. Lipid Res. 2009, 50: Suppl: S172-S177). Therefore, PCSK9 is a well-validated target for LDL-choleserol-lowering therapy (Hooper et al., Expert Opin. Biol. Ther. 2013, 13: 429-435).
Antibodies for blocking PCSK9, Alirocumab and Evolocumab, have been demonstrated to reduce circulating PCSK9 levels and lower LDL-cholesterol levels but have a short duration of action, necessitating frequent subcutaneous injections (Zhang et al., BMC Med. 2015, 13: 123;
Navarese et al., Ann. Intern. Med.
2015, 163: 40-51).
It is an object herein to provide compounds, methods, and pharmaceutical compositions for the improved treatment of diseases such as cardiovascular diseases, dyslipidemias, mixed dyslipidemias, and hypercholesterolemia.
Summary Provided herein are compounds and methods for reducing the amount or activity of PCSK9 mRNA, and in certain embodiments, reducing the amount of PCSK9 protein in a cell or animal. In certain embodiments, the animal has a cardiovascular disease. In certain embodiments, the disease is dyslipidemia. In certain embodiments, the disease is mixed dyslipidemia. In certain embodiments, the disease is hypercholesterolemia.
Certain compounds provided herein are directed to compounds and compositions that reduce LDL-cholesterol in an animal.
Certain embodiments provided herein are directed to potent and tolerable compounds and compositions useful for inhibiting PCSK9 expression, which can be useful for treating, preventing, ameliorating, or slowing progression of a cardiovascular disease. Certain embodiments provided herein are directed to compounds and compositions that are more potent or have greater therapeutic value than compounds publicly disclosed.
Detailed Description It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including"
as well as other forms, such as "includes" and "included", is not limiting.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI
reference sequence records are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Compounds described by ISIS number indicate a combination of nucleobase sequence, chemical modification, and motif.
Definitions Unless otherwise indicated, the following terms have the following meanings:
"2' -deoxynucleoside" means a nucleoside comprising 2' -H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2' -deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).
"2' -0-methoxyethyl" (also 2' -MOE and 2' -0(CH2)2-0CH3) refers to an 0-methoxy-ethyl modification at the 2' position of a furanosyl ring. A 2' -0-methoxyethyl modified sugar is a modified sugar.
"2' -MOE nucleoside" (also 2' -0-methoxyethyl nucleoside) means a nucleoside comprising a 2' -MOE
modified sugar moiety.
2 "2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside comprising a 2' -substituted or 2'-modified sugar moiety. As used herein, "2'-substituted" or "2-modified"
in reference to a sugar moiety means a sugar moiety comprising at least one 2'-substituent group other than H
or OH.
"3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3' -most nucleotide of a particular compound.
"5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5' -most nucleotide of a particular compound.
"5-methylcytosine" means a cytosine with a methyl group attached to the 5 position.
"About" means within 10% of a value. For example, if it is stated, "the compounds affected about 70% inhibition of PCSK9", it is implied that PCSK9 levels are inhibited within a range of 60% and 80%.
"Administration" or "administering" refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.
"Amelioration" refers to an improvement or lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression or severity of one or more indicators of a condition or disease. The progression or severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
"Antisense activity" means any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound to the target.
"Antisense compound" means a compound comprising an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, oligonucleotides, ribozymes, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
"Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
3 "Antisense mechanisms" are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
"Antisense oligonucleotide" means an oligonucleotide having a nucleobase sequence that is complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof.
"Bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety. "Bicyclic sugar" or "bicyclic sugar moiety" means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not comprise a furanosyl moiety.
"Branching group" means a group of atoms having at least 3 positions that are capable of forming covalent linkages to at least 3 groups. In certain embodiments, a branching group provides a plurality of reactive sites for connecting tethered ligands to an oligonucleotide via a conjugate linker and/or a cleavable moiety.
"Cell-targeting moiety" means a conjugate group or portion of a conjugate group that is capable of binding to a particular cell type or particular cell types.
"cEt" or "constrained ethyl" means a bicyclic furanosyl sugar moiety comprising a bridge connecting the 4' -carbon and the 2' -carbon, wherein the bridge has the formula: 4' -CH(CH3)-0-2'.
"cEt nucleoside" means a nucleoside comprising a cEt modified sugar moiety.
"Chemical modification" in a compound describes the substitutions or changes through chemical reaction, of any of the units in the compound relative to the original state of such unit. "Modified nucleoside"
means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase. "Modified oligonucleotide" means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
"Chemically distinct region" refers to a region of a compound that is in some way chemically different than another region of the same compound. For example, a region having 2' -0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2' -0-methoxyethyl modifications.
"Chimeric antisense compounds" means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
"Cleavable bond" means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.
4 "Cleavable moiety" means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.
"Complementary" in reference to an oligonucleotide means the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as described herein, are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (mC) and guanine (G) unless otherwise specified. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. By contrast, "fully complementary" or "100%
complementary" in reference to oligonucleotides means that such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.
"Conjugate group" means a group of atoms that is attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
"Conjugate linker" means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
"Conjugate moiety" means a group of atoms that is attached to an oligonucleotide via a conjugate linker.
"Contiguous" in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, "contiguous nucleobases"
means nucleobases that are immediately adjacent to each other in a sequence.
"Designing" or "Designed to" refer to the process of designing a compound that specifically hybridizes with a selected nucleic acid molecule.
"Differently modified" means chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE
nucleoside and an unmodified DNA nucleoside are "differently modified," even though the DNA nucleoside is unmodified. Likewise, DNA
and RNA are "differently modified," even though both are naturally-occurring unmodified nucleosides.
Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2' -0Me modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2' -0Me modified sugar and an unmodified thymine nucleobase are not differently modified.
5 "Double-stranded antisense compound" means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an oligonucleotide.
"Effective amount" means the amount of compound sufficient to effectuate a desired physiological outcome in an individual in need of the compound. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
"Efficacy" means the ability to produce a desired effect.
"Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.
"Gapmer" means an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings."
"Hybridization" means the annealing of oligonucleotides and/or nucleic acids.
While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target.
"Immediately adjacent" means there are no intervening elements between the immediately adjacent elements of the same kind (e.g. no intervening nucleobases between the immediately adjacent nucleobases).
"Individual" means a human or non-human animal selected for treatment or therapy.
"Inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
"Internucleoside linkage" means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. "Modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring, phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.
6 "Lengthened oligonucleotides" are those that have one or more additional nucleosides relative to an oligonucleotide disclosed herein, e.g. a parent oligonucleotide.
"Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.
"Linker-nucleoside" means a nucleoside that links an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of a compound. Linker-nucleosides are not considered part of the oligonucleotide portion of a compound even if they are contiguous with the oligonucleotide.
"Mismatch" or "non-complementary" means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned. For example, nucleobases including but not limited to a universal nucleobase, inosine, and hypoxanthine, are capable of hybridizing with at least one nucleobase but are still mismatched or non-complementary with respect to nucleobase to which it hybridized. As another example, a nucleobase of a first oligonucleotide that is not capable of hybridizing to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatch or non-complementary nucleobase.
"Modulating" refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating PCSK9 RNA can mean to increase or decrease the level of PCSK9 RNA
and/or PCSK9 protein in a cell, tissue, organ or organism. A "modulator" effects the change in the cell, tissue, organ or organism. For example, a PCSK9 compound can be a modulator that decreases the amount of PCSK9 RNA and/or PCSK9 protein in a cell, tissue, organ or organism.
"MOE" means methoxyethyl.
"Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.
"Motif" means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.
"Natural" or "naturally occurring" means found in nature.
"Non-bicyclic modified sugar" or "non-bicyclic modified sugar moiety" means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
"Nucleic acid" refers to molecules composed of monomeric nucleotides. A
nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.
7 "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid. As used herein a "naturally occurring nucleobase" is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A "modified nucleobase" is a naturally occurring nucleobase that is chemically modified. A
"universal base" or "universal nucleobase" is a nucleobase other than a naturally occurring nucleobase and modified nucleobase, and is capable of pairing with any nucleobase.
"Nucleobase sequence" means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage.
"Nucleoside" means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. "Modified nucleoside" means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.
"Oligomeric compound" means a compound comprising a single oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. Unless otherwise indicated, oligonucleotides consist of 8-80 linked nucleosides. "Modified oligonucleotide" means an oligonucleotide, wherein at least one sugar, nucleobase, or internucleoside linkage is modified. "Unmodified oligonucleotide" means an oligonucleotide that does not comprise any sugar, nucleobase, or internucleoside modification.
"Parent oligonucleotide" means an oligonucleotide whose sequence is used as the basis of design for more oligonucleotides of similar sequence but with different lengths, motifs, and/or chemistries. The newly designed oligonucleotides may have the same or overlapping sequence as the parent oligonucleotide.
"Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g.
intrathecal or intracerebroventricular administration.
"Pharmaceutically acceptable carrier or diluent" means any substance suitable for use in administering to an individual. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as PBS or water-for-injection.
"Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds or oligonucleotides, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
8
9 "Pharmaceutical agent" means a compound that provides a therapeutic benefit when administered to an individual.
"Pharmaceutical composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more compounds or salt thereof and a sterile aqueous solution.
"Phosphorothioate linkage" means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.
"Phosphorus moiety" means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
"Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.
"Prevent" refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely.
"Prodrug" means a compound in a form outside the body which, when administered to an individual, is metabolized to another form within the body or cells thereof. In certain embodiments, the metabolized form is the active, or more active, form of the compound (e.g., drug). Typically conversion of a prodrug within the body is facilitated by the action of an enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) present in cells or tissues, and/or by physiologic conditions.
"Reduce" means to bring down to a smaller extent, size, amount, or number.
"RefSeq No." is a unique combination of letters and numbers assigned to a sequence to indicate the sequence is for a particular target transcript (e.g., target gene). Such sequence and information about the target gene (collectively, the gene record) can be found in a genetic sequence database. Genetic sequence.databases include the NCBI Reference Sequence database, GenBank, the European Nucleotide Archive, and the DNA
Data Bank of Japan (the latter three forming the International Nucleotide Sequence Database Collaboration or INS DC).
"Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
"RNAi compound" means an antisense compound that acts, at least in part, through RISC or Ago2, but not through RNase H, to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
"Segments" are defined as smaller or sub-portions of regions within a nucleic acid.
"Side effects" means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
"Single-stranded" in reference to a compound means the compound has only one oligonucleotide.
"Self-complementary" means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligonucleotide, wherein the oligonucleotide of the compound is self-complementary, is a single-stranded compound. A single-stranded compound may be capable of binding to a complementary compound to form a duplex.
"Sites" are defined as unique nucleobase positions within a target nucleic acid.
"Specifically hybridizable" refers to an oligonucleotide having a sufficient degree of complementarity between the oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.
"Specifically inhibit" with reference to a target nucleic acid means to reduce or block expression of the target nucleic acid while exhibiting fewer, minimal, or no effects on non-target nucleic acids. Reduction does not necessarily indicate a total elimination of the target nucleic acid's expression.
"Standard cell assay" means assay(s) described in the Examples and reasonable variations thereof.
"Standard in vivo experiment" means the procedure(s) described in the Example(s) and reasonable variations thereof.
"Sugar moiety" means an unmodified sugar moiety or a modified sugar moiety.
"Unmodified sugar moiety" or "unmodified sugar" means a 2' -0H(H) furanosyl moiety, as found in RNA (an "unmodified RNA
sugar moiety"), or a 2' -H(H) moiety, as found in DNA (an "unmodified DNA
sugar moiety"). Unmodified sugar moieties have one hydrogen at each of the l', 3', and 4' positions, an oxygen at the 3' position, and two hydrogens at the 5' position. "Modified sugar moiety" or "modified sugar"
means a modified furanosyl sugar moiety or a sugar surrogate. "Modified furanosyl sugar moiety" means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2' -substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
"Sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary compounds or nucleic acids.
"PCSK9" means any nucleic acid or protein of PCSK9. "PCSK9 nucleic acid" means any nucleic acid encoding PCSK9. For example, in certain embodiments, a PCSK9 nucleic acid includes a DNA sequence encoding PCSK9, an RNA sequence transcribed from DNA encoding PCSK9 (including genomic DNA
comprising introns and exons) and an mRNA sequence encoding PCSK9. "PCSK9 mRNA" means an mRNA
encoding a PCSK9 protein. The target may be referred to in either upper or lower case.
"PCSK9 specific inhibitor" refers to any agent capable of specifically inhibiting PCSK9 RNA and/or PCSK9 protein expression or activity at the molecular level. For example, PCSK9 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of PCSK9 RNA and/or PCSK9 protein.
"Target gene" refers to a gene encoding a target.
"Targeting" means the specific hybridization of a compound to a target nucleic acid in order to induce a desired effect.
"Target nucleic acid," "target RNA," "target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by compounds described herein.
"Target region" means a portion of a target nucleic acid to which one or more compounds is targeted.
"Target segment" means the sequence of nucleotides of a target nucleic acid to which a compound is targeted. "5' target site" refers to the 5' -most nucleotide of a target segment. "3' target site" refers to the 3'-most nucleotide of a target segment.
"Terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
"Therapeutically effective amount" means an amount of a compound, pharmaceutical agent, or composition that provides a therapeutic benefit to an individual.
"Treat" refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.

Certain Embodiments Certain embodiments provide methods, compounds and compositions for inhibiting expression.
Certain embodiments provide compounds targeted to a PCSK9 nucleic acid. In certain embodiments, .. the PCSK9 nucleic acid has the sequence set forth in RefSeq or GENBANK
Accession No. NM_174936.3 and GENBANK Accession No. NC_000001.11 truncated from nucleotides 55036001 to 55068000 (incorporated by reference, disclosed herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively). In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.
Certain embodiments provide a compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide 9 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 9 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain .. embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 10 to linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide
10 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense 25 compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide
11 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 11 contiguous nucleobases of any 30 .. of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 11 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide
12 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 12 to 30 linked nucleosides in length.
In certain embodiments, the compound comprises a modified oligonucleotide 30 linked nucleosides in length. In certain embodiments, the compound is an antisense compound or oligomeric compound.
Certain embodiments provide a compound comprising a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 14 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.
Certain embodiments provide a compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length and complementary within nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 of SEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100%
complementary to SEQ ID NO: 2. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 of SEQ ID
NO: 2, wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100%
complementary to SEQ ID NO:
2. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
In certain embodiments, compounds target a region of a PCSK9 nucleic acid. In certain embodiments, such compounds targeted to a region of a PCSK9 nucleic acid have a contiguous nucleobase portion that is complementary to an equal length nucleobase portion of the region. For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobase portion complementary to an equal
13 length portion of a region recited herein. In certain embodiments, such compounds target the following nucleotide regions of SEQ ID NO: 2: 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136. In certain embodiments, these compounds are antisense compounds, oligomeric compounds, or oligonucleotides.
In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 6356-6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 of SEQ ID NO: 2.
In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and complementary within nucleotides 6356-6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 of SEQ ID
NO: 2. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs:
1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.
In certain embodiments, compounds targeted to PCSK9 are ISIS 863568, ISIS
863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS
863681. Out of over 1540 compounds that were screened as described in the Examples section below, ISIS
863568, ISIS 863579, ISIS
863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 emerged as the top lead compounds. In particular, ISIS 863633 exhibited the best combination of properties in terms of potency and tolerability out of over 1540 compounds.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified sugar. In certain embodiments, at least one modified sugar comprises a 2' -0-methoxyethyl group. In certain embodiments, at least one modified sugar is a bicyclic sugar, such as a 4'-CH(CH3)-0-2' group, a 4'-CH2-0-2' group, or a 4' -(CH2)2-0-2' group.
14 In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.
In certain embodiments, any of the foregoing modified oligonucleotides comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide is 14 to 80 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, the modified oligonucleotide is 10 to 30 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, the modified oligonucleotide is 14 linked nucleosides in length having a nucleobase sequence consisting of the sequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.
In certain embodiments, a compound comprises or consists of a modified oligonucleotide 16-80 linked nucleobases in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID
NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of three linked nucleosides; and a 3' wing segment consisting of three linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide consists of 16-30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.
In certain embodiments, a compound comprises or consists of ISIS 863633 or salt thereof, having the following chemical structure:

OH N2ek,N, HO OH 0 0-p=0 I ILN
- Rc, 0, N N
N...-1.0 0"---TrN'll'' HN
HO - (j) H 0 N1,NH N0N l' "14 NH2 Y ) =--1.2"\''' HS-P=0 N2t*N HS-1=0 NII..-N
HO 0---TrN

NH \ N N 0 (LY ---(-)/N N

HO OH I- ---, 0 IL

HS-P=0 A'NH
rThrs'N"-kjo HS-p=0 --k-HO C 4 H A).(NH 0 IciI/N 0 ....Tr-NH \ N0 (')//-NII)I:r NH2 HS-p=0 HS-p=0 N2e.:"N 0---vl_)/N N NH2 HS-p=0 HS-p=0 Na. N
µµµ()/

N
'(*N
0 HS-P=0 I

HS-p=0 '1(.11'Ir 03(:)/

N.--0 NH2 HS-P=0 NIA-N

HS-P=0 N 0 N N

C')/ 0 )1 HS-p=0 <NIA2X.1 HS-p=0 NI.1r 0 >_ N

OH
HS p, 0 _____ In certain embodiments, a compound comprises or consists of ISIS 863633 or salt thereof, having the following chemical structure:

9 Nx-L, N
HO OH CLI N
_......r.O...\, HNRc 0"---Tr 4 H 0 (-JO
_______________________________________________________________ (:)_VN 0 HO
NH 'N

HO OH 0 0 N 0 N e Nx-L....N
HO
....r.2..\.,0(-rN ep-ro a*N s1-0 I
---' OV \

'..1 HO_. ,....r2._\ e i ik' 0-----r 1 N 0 S-p=0 Ow =-kb 4 H 1(1\L1H
s-17-0 ANH
NH 0\

0,/ N --'0 CD Niii-NH
NH2 s->=0 I ,L
os-7=0 Nx-L.N 0.N

S-p=0 tIl NH2 o es17=o Nx--1:-...N
_CLyN 0 c_o_V e9 N
0 S+0 I _t e 9 0 - -.
s-c>=0 ill'ir o.., N--.0 _CL), NH2 e ' NH2 S-P=0 e 0 1 I
s+0 N 0 N
N
0 I _t, N¨'0 _0_)/

e 9 0 , S-P=0 1\11r S-p=0 A)L'I\t1H
N N
N (1)/N NH2 N-'.0 c5/ 0 0? OH
S p 0 ___________________________________________________________ In certain embodiments, a compound comprises or consists of the sodium salt of ISIS 863633, having the following chemical structure:

N
abbe N? N _o_vN,120 T , HO OH 0 01'=0 a\I NeN
0 HNRCb 0, N N
_......r.L.\,) CrIr N
HO 4 H c = ',...10 ..)( O 1'o '70 NH N
HO OH 0 N 0 e 9 Nx-1,..N
es-Fro NI.,-"L.N
S-P=0 I
4 H 0 0\ I 0 1 Na 0 N N
NH OV Na I4;//N N
'---C)-1 -'ki o o o o HO OH e 9 S-P=0 _, e i 0 , S-P=0Nallo HO 0---trN 0 4 H 0 1 il(NH W 0 Na 0 NH \

'N'L0 0 9 o NIANH
/-o----0 NH2 S-P=0 I
0 , es-7=o NIA,..N Na 0..

0 e 9 Na0 I 0 ¨c)¨Y
e sl7=o tX
9 NH2 Na 0 e eS-P=0 Nx-L, N
\-2-)//N 0 Na I
N N

0 e N
S-P=t 9e 9- 9 0 I
_ 1 Na S-F,' 111'11H Na 0 N "
/

' NH2 S-P=0 Nf....N
Na e e 9 0 1 I
e S4,7=0 N 0 N N
Na 0 I

-=0 1\11,11:IH
Na S-F.0 SP
ILIIIH 0 ,I, Na N

c-L/N

0?
o OSPO _____________________________________________________________________ OH
Na "

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding PCSK9.
In any of the foregoing embodiments, the compound can be single-stranded. In certain embodiments, the compound comprises deoxyribonucleotides. In certain embodiments, the compound is double-stranded. In certain embodiments, the compound is double-stranded and comprises ribonucleotides. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
In any of the foregoing embodiments, the compound can be 8 to 80, 10 to 30, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked nucleosides in length.
In certain embodiments, the compound comprises or consists of an oligonucleotide.
In certain embodiments, a compound comprises a modified oligonucleotide described herein and a conjugate group. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N- Acetylgalactosamine (GalNAc), at least two N-Acetylgalactosamines (GalNAcs), or at least three N- Acetylgalactosamines (GalNAcs).
In certain embodiments, compounds or compositions provided herein comprise a pharmaceutically acceptable salt of the modified oligonucleotide. In certain embodiments, the salt is a sodium salt. In certain embodiments, the salt is a potassium salt.
In certain embodiments, the compounds or compositions as described herein are active by virtue of having at least one of an in vitro IC50 of less than 5 M, less than 4.5 M, less than 4 M, less than 3.5 M, less than 3 M, less than 2.5 M, less than 2 M, lless than 1.5 M, less than 1 M, less than 0.9 M, less than 0.8 M, less than 0.7 M, less than 0.6 M, less than 0.5 M, less than 0.4 M, less than 0.3 M, less than 0.2 M, or less than 0.1 M.
In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase an alanine transaminase (ALT) or aspartate transaminase (AST) value of no more than 4 fold, 3 fold, or 2 fold over saline treated animals or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2% compared to control treated animals.
In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over control treated animals.
In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over control animals.
Certain embodiments provide a composition comprising the compound of any of the aforementioned .. embodiments or any pharmaceutically acceptable salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain embodiments, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain embodiments, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature or about 20 C, about 21 C, about 22 C, about 23 C, about 24 C, about 25 C, about 26 C, about 27 C, about 28 C, about 29 C, or about C.
30 Certain Indications Certain embodiments provided herein relate to methods of inhibiting PCSK9 expression, which can be useful for treating, preventing, or ameliorating a disease associated with PCSK9 in an individual, by administration of a compound that targets PCSK9. In certain embodiments, the compound can be a PCSK9 specific inhibitor. In certain embodiments, the compound can be an antisense compound, oligomeric compound, or oligonucleotide targeted to PCSK9.

Examples of diseases associated with PCSK9 treatable, preventable, and/or ameliorable with the methods provided herein include a cardiovascular disease, dyslipidemia, mixed dyslipidemia, hypercholesterolemia, a reduction in LDL cholesterol, and reduction in atherogenic apolipoprotein (a) [Lp(a)].
In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with PCSK9 in an individual comprises administering to the individual a compound comprising a PCSK9 specific inhibitor, thereby treating, preventing, or ameliorating the disease. In certain embodiments, the individual is identified as having or at risk of having a disease associated with PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ
ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally.
In certain embodiments, administering the compound reduces LDL-cholesterol levels, reduces Lp(a) levels, induces LDL receptor (LDL-R) activity, and regulates LDL receptor-LDL-cholesterol homeostasis.
In certain embodiments, a method of treating, preventing, or ameliorating a cardiovascular disease comprises administering to the individual a compound comprising a PCSK9 specific inhibitor, thereby treating, preventing, or ameliorating the cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS
863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681.
In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound reduces LDL-cholesterol levels. In certain embodiments, administering the compound reduces Lp(a) levels. In certain embodiments, administering the compound induces LDL receptor (LDL-R) activity.
In certain embodiments, administering the compound regulates LDL receptor-LDL-cholesterol homeostasis.
In certain embodiments, a method of inhibiting expression of PCSK9 in an individual having, or at risk of having, a disease associated with PCSK9 comprises administering to the individual a compound comprising a PCSK9 specific inhibitor, thereby inhibiting expression of PCSK9 in the individual. In certain embodiments, administering the compound inhibits expression of PCSK9 in the liver. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the individual has, or is at risk of having dyslipidemia. In certain embodiments, the individual has, or is at risk of having hypercholesterolemia. In certain embodiments, the individual has, or is at risk of having mixed dyslipidemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS

863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681.
In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound reduces LDL-cholesterol levels. In certain embodiments, administering the compound reduces Lp(a) levels. In certain embodiments, administering the compound induces LDL receptor (LDL-R) activity.
In certain embodiments, administering the compound regulates LDL receptor-LDL-cholesterol homeostasis.
In certain embodiments, a method of inhibiting expression of PCSK9 in a cell comprises contacting the cell with a compound comprising a PCSK9 specific inhibitor, thereby inhibiting expression of PCSK9 in the cell. In certain embodiments, the cell is a hepatocyte. In certain embodiments, the cell is in the liver. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having a cardiovascular disease. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having dyslipidemia. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having mixed dyslipidemia. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having hypercholesterolemia. In certain embodiments, administering the compound inhibits expression of PCSK9 in the liver. In certain embodiments, the individual has, or is at risk of having, a cardiovascular disease.
In certain embodiments, the individual has, or is at risk of having dyslipidemia. In certain embodiments, the individual has, or is at risk of having mixed dyslipidemia. In certain embodiments, the individual has, or is at risk of having hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9.
In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID
NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS
863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681.
In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.

In certain embodiments, a method of reducing or inhibiting LDL-cholesterol levels, reducing or inhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, or regulating LDL receptor-LDL-cholesterol homeostasis in the liver of an individual having, or at risk of having, a disease associated with PCSK9 comprises administering to the individual a compound comprising a PCSK9 specific inhibitor, thereby reducing or inhibiting LDL-cholesterol and Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL
receptor-LDL-cholesterol homeostasis in the liver of the individual. In certain embodiments, the individual has, or is at risk of having, a cardiovascular disease. In certain embodiments, the individual has, or is at risk of having, dyslipidemia. In certain embodiments, the individual has, or is at risk of having, mixed dyslipidemia.
In certain embodiments, the individual has, or is at risk of having, hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9.
In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540.
In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ
ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally.
In certain embodiments, the individual is identified as having or at risk of having a disease associated with PCSK9.
Certain embodiments are drawn to a compound comprising a PCSK9 specific inhibitor for use in treating a disease associated with PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the disease is dyslipidemia. In certain embodiments, the disease is mixed dyslipidemia.
In certain embodiments, the disease is hypercholesterolemia In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS
863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681.
In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally.
Certain embodiments are drawn to a compound comprising a PCSK9 specific inhibitor for use in reducing or inhibiting LDL-cholesterol, reducing or inhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL receptor-LDL-cholesterol homeostasis of an individual having or at risk of having a cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ
ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.

Certain embodiments are drawn to use of a compound comprising a PCSK9 specific inhibitor for the manufacture or preparation of a medicament for treating a disease associated with PCSK9. Certain embodiments are drawn to use of a compound comprising a PCSK9 specific inhibitor for the preparation of a medicament for treating a disease associated with PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the disease is dyslipidemia.
In certain embodiments, the disease is mixed dyslipidemia. In certain embodiments, the disease is hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9.
In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540.
In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ
ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
Certain embodiments are drawn to use of a compound comprising a PCSK9 specific inhibitor for the manufacture or preparation of a medicament for reducing or inhibiting LDL-cholesterol levels, reducing or inhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL receptor-LDL-cholesterol homeostasis of an individual having or at risk of having a cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. Certain embodiments are drawn to use of a compound comprising a PCSK9 specific inhibitor for the preparation of a medicament for reducing or inhibiting LDL-cholesterol levels, reducing or inhibiting Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL receptor-LDL-cholesterol homeostasis of an individual having or at risk of having a cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound comprises an antisense compound targeted to PCSK9. In certain embodiments, the compound comprises an oligonucleotide targeted to PCSK9. In certain embodiments, a compound comprises a modified oligonucleotide 8 to 80 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 3-1540. In certain embodiments, a compound comprises a modified oligonucleotide 14 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ
ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353. In any of the foregoing embodiments, the modified oligonucleotide can be 10 to 30 linked nucleosides in length. In certain embodiments, the compound is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
In any of the foregoing methods or uses, the compound can be targeted to PCSK9. In certain embodiments, the compound comprises or consists of a modified oligonucleotide, for example a modified oligonucleotide 8 to 80 linked nucleosides in length, 10 to 30 linked nucleosides in length, 12 to 30 linked nucleosides in length, or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, 85%, 90%, 95% or 100% complementary to any of the nucleobase sequences recited in SEQ
ID NOs: 1, 2, 1545-1550. In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage, the modified sugar is a bicyclic sugar or a 2' -0-methoxyethyl, and the modified nucleobase is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In any of the foregoing embodiments, the modified oligonucleotide is 12 to 30,
15 to 30, 15 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 19 to 22, 20 to 22,
16 to 20, or 17 or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, 85%, 90%, 95% or 100%
complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1, 2, 1545-1550. In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage, the modified sugar is a bicyclic sugar or a 2' -0-methoxyethyl, and the modified nucleobase is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked 2' -deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In any of the foregoing methods or uses, the compound comprises or consists of a modified oligonucleotide 14 to 30 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs: 3-1540, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked 2'-deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In any of the foregoing methods or uses, the compound comprises or consists a modified oligonucleotide 16 to 80 linked nucleobases in length having a nucleobase sequence comprising or consisting of the sequence recited in any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of three linked nucleosides; and a 3' wing segment consisting of three linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar;
wherein each internucleoside linkage is a phosphorothioate linkage; wherein each cytosine is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide is 16-80 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is 16-30 linked nucleosides in length.
In any of the foregoing methods or uses, the compound comprises or consists of ISIS 863633 or salt thereof, having the following chemical structure:

OH N2ek,N, HO OH 0 0-p=0 I ILN
HNRc, 0, N
N...-1.0 0"---Tr'N'IL' N1, N, O N l' "14N

Y ) =--1.2"\''' HS-P=0 I N HS-p=0 NII..-N
H
HO 0---TrN

0 \
N N
NH
OV ;1-2-Y

HO OH õ.õ 0 HS-P=0 A'ILNH
CrThrs'N"-kjo HS-p=0 --k-HO 4 H A).(NH 0 IciIy/N NO
....Tr- 0 0 NH 0\

(--4//
NI1-11:r T NH2 HS-p=0 HS-p=0 N2e.:"N 0---vl_)/N N NH2 HS-p=0 HS-p=0 Na. N
µµµ()/

N
ILN
0 HS-P=0 9 'N...kb HS-p=0 '1(.11'Ir 03(:)/
N.--NH2 HS-P=0 NIA-N

HS-P=0 T:LN 0 N N

C')/ 0 Y )1 HS-p=0 <NIA2X.1 HS-p=0 NI.1r N''0 0 0 >_ N
c-22/ 0 OH
HS p, 0 _________________________________________________________ o In any of the foregoing methods or uses, the compound comprises or consists of ISIS 863633 or salt thereof, having the following chemical structure:

ne NH2 NH2 Y Nxi,, N
HO OH CLI N
_......r.O..\, HNRc 0"---TrN"-k--HO
NH 'N

e 9 Nx-L...N
HO
._....1.2...\., )1õ,oõ_,--NH es-Fro NIA.:- N S-p=0 I
0"---'trN

OV \

....1 HO OH e 0 HO_ ,c) .ik'....r2...\ i .1),,c 0 S-p=0 1 S+0 iNH
e NH Ow =-kb Nr.NH 0\

0,/ N --'0 0 9 NiKNH
NH2 s-P=0 I ,L
es-0 Nx-L.N 0.N

\)-2-)/I e 9 s-F,7=o 'N(I'XI
9 NH2 o Os_p=0 Nx-LN, N

I

e9 N
0 S4,7=0 I
S-1,=0=0 0 9 ill' Ir N 0 0-..õ
N.--0 e , NH2 S-P=0 Nfz,N
e9 1 I
s+0 N 0 N N

N.,.
_0_)/N 0 0 9 0 , S-P=0 1\11r S-p=0 1\r c5/ 0 0? OH
S p, 0 __________________________________________________________ In any of the foregoing methods or uses, the compound comprises or consists of the sodium salt of ISIS
863633, having the following chemical structure:

NH, ne N?
_o_vN1120 T N , HO OH 0 0-1:,'=0 h NeN
0 HNRCb 0, N N
_......r.L.\,) CrIr N
HO 4 H c = \ 10 ON
..)(NH N 1'o ,-0177-0 HO OH 0 0 0 Nx-L.,N
HO._ 0----0S-7=0 (NN S-P=0 I
tr N
4 H 0 0\ e I , Na 0 N N
NH OV Na I/N N
'---C)-1 HO OH e 9 S-F'=0 i Na , S-P=0 I
HO 0.--/rN 0 4 H 0 1 ill'NH W 0 Na O\NH
0 () 'N'L0 I(NH

9 NH2 S-P=0 0s-7=o NIA,..N Na 0N N NH2 Na0 0 I 0 e (?
e s-o NH2 Na 0 e Na I
eS-P=0 Nx-L, N
\ NH2 -2-)//N 0 N N
O e N
0 Na S-F0 S-P=0 I
I-; I
@ 1 N 0 O--.
Na ' NH2 :s-=o Nf....N
e 0 1 I
e s+c) N Na 0 N N
Na 0 I
_0_)/N 0 e 9 0 , F S-P=0 1\11,11:IH
Na0 S-,'.0 Ajj11H 0 Is Na N

0?
o OSPO ____________ OH
Na "

In any of the foregoing methods or uses, the compound can be administered parenterally. For example, in certain embodiments the compound can be administered through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g.
intrathecal or intracerebroventricular administration.
Certain Compounds In certain embodiments, compounds described herein can be antisense compounds.
In certain embodiments, the antisense compound comprises or consists of an oligomeric compound. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.
In certain embodiments, a compound described herein comprises or consists of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.

In certain embodiments, a compound or antisense compound is single-stranded.
Such a single-stranded compound or antisense compound comprises or consists of an oligomeric compound. In certain embodiments, such an oligomeric compound comprises or consists of an oligonucleotide and optionally a conjugate group. In certain embodiments, the oligonucleotide is an antisense oligonucleotide. In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a single-stranded antisense compound or oligomeric compound comprises a self-complementary nucleobase sequence.
In certain embodiments, compounds are double-stranded. Such double-stranded compounds comprise a first modified oligonucleotide having a region complementary to a target nucleic acid and a second modified oligonucleotide having a region complementary to the first modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an RNA oligonucleotide. In such embodiments, the thymine nucleobase in the modified oligonucleotide is replaced by a uracil nucleobase. In certain embodiments, compound comprises a conjugate group. In certain embodiments, one of the modified oligonucleotides is conjugated. In certain embodiments, both the modified oligonucleotides are conjugated. In certain embodiments, the first modified oligonucleotide is conjugated. In certain embodiments, the second modified oligonucleotide is conjugated. In certain embodiments, the first modified oligonucleotide is 12-30 linked nucleosides in length and the second modified oligonucleotide is 12-30 linked nucleosides in length. In certain embodiments, one of the modified oligonucleotides has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of SEQ ID
NOs: 3-1540.
In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such double-stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group. The oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.
Examples of single-stranded and double-stranded compounds include but are not limited to oligonucleotides, siRNAs, microRNA targeting oligonucleotides, and single-stranded RNAi compounds, such as small hairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNA
mimics.
In certain embodiments, a compound described herein has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, a compound described herein comprises an oligonucleotide 10 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 22 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 30 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 30 linked subunits in length.
In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 21 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 20 linked subunits in length. In certain embodiments, a compound 15 described herein comprises an oligonucleotide 20 to 30 linked subunits in length. In other words, such oligonucleotides are 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits in length, respectively. In certain embodiments, a compound described herein comprises an oligonucleotide 14 linked subunits in length. In 20 certain embodiments, a compound described herein comprises an oligonucleotide 16 linked subunits in length.
In certain embodiments, a compound described herein comprises an oligonucleotide 17 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 18 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 19 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 20 linked subunits in length. In other embodiments, a compound described herein comprises an oligonucleotide 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the compound described herein comprises an oligonucleotide 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the linked subunits are nucleotides, nucleosides, or nucleobases.
In certain embodiments, the compound may further comprise additional features or elements, such as a conjugate group, that are attached to the oligonucleotide. In certain embodiments, such compounds are antisense compounds. In certain embodiments, such compounds are oligomeric compounds. In embodiments where a conjugate group comprises a nucleoside (i.e. a nucleoside that links the conjugate group to the oligonucleotide), the nucleoside of the conjugate group is not counted in the length of the oligonucleotide.
In certain embodiments, compounds may be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated compound targeted to a PCSK9 nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the compound.
Alternatively, the deleted nucleosides may be dispersed throughout the compound.
When a single additional subunit is present in a lengthened compound, the additional subunit may be located at the 5' or 3' end of the compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in a compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the compound.
Alternatively, the added subunits may be dispersed throughout the compound.
It is possible to increase or decrease the length of a compound, such as an oligonucleotide, and/or introduce mismatch bases without eliminating activity (Woolf et al. Proc.
Natl. Acad. Sci. USA 1992, 89:7305-7309; Gautschi etal. J. Natl. Cancer Inst. March 2001, 93:463-471; Maher and Dolnick Nuc. Acid. Res. 1998, 16:3341-3358). However, seemingly small changes in oligonucleotide sequence, chemistry and motif can make large differences in one or more of the many properties required for clinical development (Seth et al. J. Med.
Chem. 2009, 52, 10; Egli et al. J. Am. Chem. Soc. 2011, 133, 16642).
In certain embodiments, compounds described herein are interfering RNA
compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term "RNAi" is meant to be equivalent to other terms used to describe sequence specific RNA
interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PCSK9 described herein. In certain embodiments, the compound can be double-stranded.
In certain embodiments, the compound comprises a first strand comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobase portion of any one of SEQ ID
NOs: 3-1540 and a second strand.
In certain embodiments, the compound comprises a first strand comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540 and a second strand. In certain embodiments, the compound comprises ribonucleotides in which the first strand has uracil (U) in place of thymine (T) in any one of SEQ ID NOs: 3-1540. In certain embodiments, the compound comprises (i) a first strand comprising a nucleobase sequence complementary to the site on PCSK9 to which any of SEQ ID NOs: 3-1540 is targeted, and (ii) a second strand. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group; 2'-F) or contains an alkoxy group (such as a methoxy group; 2'-OMe). In certain embodiments, the compound comprises at least one 2'-F sugar modification and at least one 2' -0Me sugar modification. In certain embodiments, the at least one 2' -F
sugar modification and at least one 2'-0Me sugar modification are arranged in an alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along a strand of the dsRNA compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the compound contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000.
In certain embodiments, the first strand of the compound is an siRNA guide strand and the second strand of the compound is an siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, each strand of the compound is 16,
17, 18, 19, 20, 21, 22, or 23 linked nucleosides in length. In certain embodiments, the first or second strand of the compound can comprise a conjugate group.
In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PCSK9 described herein. In certain embodiments, the compound is single stranded. In certain embodiments, such a compound is a single-stranded RNAi (ssRNAi) compound. In certain embodiments, the compound comprises at least an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobase portion of any one of SEQ ID NOs: 3-1540. In certain embodiments, the compound comprises the nucleobase sequence of any one of SEQ ID NOs: 3-1540 In certain embodiments, the compound comprises ribonucleotides in which uracil (U) is in place of thymine (T) in any one of SEQ ID NOs: 3-1540. In certain embodiments, the compound comprises a nucleobase sequence complementary to the site on PCSK9 to which any of SEQ ID NOs: 3-1540 is targeted. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group; 2' -F) or contains an alkoxy group (such as a methoxy group; 2'-0Me). In certain embodiments, the compound comprises at least one 2'-F sugar modification and at least one 2' -0Me sugar modification. In certain embodiments, the at least one 2'-F sugar modification and at least one 2' -0Me sugar modification are arranged in an alternating pattern for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along a strand of the compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat.
No. 6,673,661. In other embodiments, the compound contains a capped strand, as disclosed, for example, by WO 00/63364, filed Apr.
19, 2000. In certain embodiments, the compound consists of 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides.
In certain embodiments, the compound can comprise a conjugate group.
In certain embodiments, compounds described herein comprise modified oligonucleotides. Certain modified oligonucleotides have one or more asymmetric center and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or 13 such as for sugar anomers, or as (D) or (L) such as for amino acids etc.
Included in the modified oligonucleotides provided herein are all such possible isomers, including their racemic and optically pure forms, unless specified otherwise. Likewise, all cis- and trans-isomers and tautomeric forms are also included.
The compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 1H hydrogen atoms.
Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2H or 3H in place of 1H, 13C or 14C in place of 12C, 15N in place of 'N, 170 or 180 in place of 160, and 33S, 34S, 35S, or 36S in place of 325. In certain embodiments, non-radioactive isotopic substitutions may impart new properties on the compound that are beneficial for use as a therapeutic or research tool. In certain embodiments, radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes, such as an imaging assay.
Certain Mechanisms In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. In certain embodiments, compounds described herein are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, compounds described herein selectively affect one or more target nucleic acid. Such compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in a significant undesired antisense activity.
In certain antisense activities, hybridization of a compound described herein to a target nucleic acid results in recruitment of a protein that cleaves the target nucleic acid. For example, certain compounds described herein result in RNase H mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, compounds described herein are sufficiently "DNA-like" to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.
In certain antisense activities, compounds described herein or a portion of the compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain compounds described herein result in cleavage of the target nucleic acid by Argonaute.
Compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).
In certain embodiments, hybridization of compounds described herein to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid.
In certain such embodiments, hybridization of the compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of the compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of the compound to a target nucleic acid results in alteration of translation of the target nucleic acid.
Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.
Target Nucleic Acids, Target Regions and Nucleotide Sequences In certain embodiments, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA
and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.
Nucleotide sequences that encode PCSK9 include, without limitation, the following: RefSEQ No.
GENBANK Accession No. NM_174936.3 (SEQ ID NO: 1), GENBANK Accession No.
NC_000001.11 truncated from nucleotides 55036001 to 55068000 (SEQ ID NO: 2), GENBANK
Accession No. AK124635.1 (SEQ ID NO: 1545), GENBANK Accession No. NT_032977.8 truncated from nucleotides 25475000 to 25504000 (SEQ ID NO: 1546), GENBANK Accession No. DA092236.1 (SEQ ID NO:
1547), GENBANK
Accession No. DA803830.1 (SEQ ID NO: 1548), GENBANK Accession No. DC352135.1 (SEQ ID NO:
1549), and GENBANK Accession No. NR_110451.1 (SEQ ID NO: 1550).

Hybridization In some embodiments, hybridization occurs between a compound disclosed herein and a PCSK9 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Hybridization conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the compounds provided herein are specifically hybridizable with a PCSK9 nucleic acid.
Complementarity An oligonucleotide is said to be complementary to another nucleic acid when the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as described herein, are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methyl cytosine (mC) and guanine (G) unless otherwise specified.
Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. An oligonucleotide is fully complementary or 100%
complementary when such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.
In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. Non-complementary nucleobases between a compound and a PCSK9 nucleic acid may be tolerated provided that the compound remains able to specifically hybridize to a target nucleic acid. Moreover, a compound may hybridize over one or more segments of a PCSK9 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the compounds provided herein, or a specified portion thereof, are, are at least, or are up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a PCSK9 nucleic acid, a target region, target segment, or specified portion thereof. In certain embodiments, the compounds provided herein, or a specified portion thereof, are 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, or any number in between these ranges, complementary to a PCSK9 nucleic acid, a target region, target segment, or specified portion thereof.
Percent complementarity of a compound with a target nucleic acid can be determined using routine methods.

For example, a compound in which 18 of 20 nucleobases of the compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, a compound which is 18 nucleobases in length having four non-complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid. Percent complementarity of a compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, compounds described herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, a compound may be fully complementary to a PCSK9 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of a compound is complementary to the corresponding nucleobase of a target nucleic acid. For example, a 20 nucleobase compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the compound.
Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase compound is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the compound. At the same time, the entire 30 nucleobase compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the compound are also complementary to the target sequence.
In certain embodiments, compounds described herein comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain such embodiments selectivity of the compound is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5' -end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3' -end of the gap region.
In certain such embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5' -end of the wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3' -end of the wing region. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide not having a gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5' -end of the oligonucleotide. In certain such embodiments, the mismatch is at position , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3' -end of the oligonucleotide.
The location of a non-complementary nucleobase may be at the 5' end or 3' end of the compound.
Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e.
linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer oligonucleotide.
In certain embodiments, compounds described herein that are, or are up to 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PCSK9 nucleic acid, or specified portion thereof.
In certain embodiments, compounds described herein that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PCSK9 nucleic acid, or specified portion thereof.
In certain embodiments, compounds described herein also include those which are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e.
linked) nucleobases within a region or segment of a target nucleic acid. A
"portion" can also refer to a defined number of contiguous nucleobases of a compound. In certain embodiments, the-compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 13 nucleobase portion of a target segment.
In certain embodiments, the compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 15 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 16 nucleobase portion of a target segment. Also contemplated are compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity The compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof. In certain embodiments, compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, compounds described herein are modified oligonucleotides. As used herein, a compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA
which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the compounds described herein as well as compounds having non-identical bases relative to the compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the compound. Percent identity of an compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, compounds described herein, or portions thereof, are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 A or 100%
identical to one or more of the compounds or SEQ ID NOs, or a portion thereof, disclosed herein. In certain embodiments, compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identical, or any percentage between such values, to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof, in which the compounds comprise an oligonucleotide having one or more mismatched nucleobases. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5' -end of the oligonucleotide. In certain such embodiments, the mismatch is at position , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3' -end of the oligonucleotide.
In certain embodiments, compounds described herein comprise or consist of antisense compounds. In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, compounds described herein comprise or consist of oligonucleotides. In certain embodiments, a portion of the oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Certain Modified Compounds In certain embodiments, compounds described herein comprise or consist of oligonucleotides consisting of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).
A. Modified Nucleosides Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modifed sugar moiety and a modified nucleobase.
1. Modified Sugar Moieties In certain embodiments, sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties.
In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more acyclic substituent, including but not limited to substituents at the 2', 4', and/or 5' positions. In certain embodiments one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2' -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2' -F, 2'-0CH3("0Me" or "0-methyl"), and 2'-0(CH2)20CH3_("MOE"). In certain embodiments, 2' -substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, 0-C1-C10 alkoxy, 0-C1-C10 substituted alkoxy, 0-C1-C10 alkyl, 0-C1-C10 substituted alkyl, 5-alkyl, N(R.)-alkyl, 0-alkenyl, S-alkenyl, N(R.)-alkenyl, 0-alkynyl, 5-alkynyl, N(R.)-alkynyl, 0-alkyleny1-0-alkyl, alkynyl, alkaryl, aralkyl, 0-alkaryl, 0-aralkyl, 0(CH2)25CH3, 0(CH2)20N(R.)(RO
or OCH2C(=0)-N(R.)(R.), where each R. and R. is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, and the 2' -substituent groups described in Cook et al., U.S.
6,531,584; Cook et al., U.S. 5,859,221; and Cook et al., U.S. 6,005,087. Certain embodiments of these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4'-substituent groups suitable for linearly non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO
2015/106128. Examples of 5' -substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5' -methyl (R or S), 5'-vinyl, and 5' -methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., U52010/190837 and Rajeev et al., U52013/0203836.
In certain embodiments, a 2' -substituted nucleoside or 2'- non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2' -substituent group selected from: F, NH2, N3, OCF3, OCH3, 0(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH2CH2OCH3, 0(CH2)25CH3, 0(CH2)20N(R.)(R.), 0(CH2)20(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(=0)-N(R.)(R.)), where each R. and R. is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.

In certain embodiments, a 2' -substituted nucleoside or 2'- non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2' -substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, 0(CH2)2SCH3, 0(CH2)20N(CH3)2, 0(CH2)20(CH2)2N(CH3)2, and OCH2C(=0)-N(H)CH3 ("NMA").
In certain embodiments, a 2' -substituted nucleoside or 2'- non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2' -substituent group selected from: F, OCH3, and OCH2CH2OCH3.
Nucleosides comprising modified sugar moieties, such as non-bicyclic modified sugar moieties, are referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside. For example, nucleosides comprising 2'-substituted or 2-modified sugar moieties are referred to as 2'-substituted nucleosides or 2-modified nucleosides.
Certain modifed sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' furanose ring atoms. Examples of such 4' to 2' bridging sugar substituents include but are not limited to: 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2' ("LNA"), 4'-CH2-S-2', 4'-(CH2)2-0-2' ("ENA"), 4'-CH(CH3)-0-2' (referred to as "constrained ethyl" or "cEt" when in the S configuration), 4' -CH2-0-CH2-2' , 4'-CH2-N(R)-2', 4'-CH(CH2OCH3)-0-2' ("constrained MOE" or "cM0E") and analogs thereof (see, e.g., Seth et al., U.S. 7,399,845, Bhat et al., U.S. 7,569,686, Swayze et al., U.S. 7,741,457, and Swayze et al., U.S. 8,022,193), 4'-C(CH3)(CH3)-0-2' and analogs thereof (see, e.g., Seth et al., U.S. 8,278,283), 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., Prakash et al., U.S. 8,278,425), 4'-CH2-0-N(CH3)-2' (see, e.g., Allerson et al., U.S. 7,696,345 and Allerson et al., U.S. 8,124,745), 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou, et al., J. Org. Chem.,2009, 74, 118-134), 4'-CH2-C(=CH2)-2' and analogs thereof (see e.g.õ Seth et al., U.S.
8,278,426), 4' -C(RaRb)-N(R)-0-2' , 4' -C(RaRb)-0-N(R)-2' , 4'-CH2-0-N(R)-2', and 4'-CH2-N(R)-0-2', wherein each R, Ra, and Rb is, independently, H, a protecting group, or CI-Cu alkyl (see, e.g. Imanishi et al., U.S.
7,427,672).
In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from: 4C(Ra)(Rb)].-, 4C(Ra)(Rb)].-0-, -C(Ra)=C(Rb)-, -C(Ra)N, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -S(Ra)2, -S(=0)-, and -N(Ra)-;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, CI-Cu alkyl, substituted CI-Cu alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, Cs-Cm aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, 0J1, NJ1J2, SJi, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-Ji); and each Ji and J2 is, independently, H, Ci-C12 alkyl, substituted CI-Cu alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, CI-Cu aminoalkyl, substituted CI-Cu aminoalkyl, or a protecting group.
Additional bicyclic sugar moieties are known in the art, see, for example:
Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;
Wahlestedt et al., Proc. Natl.
Acad. Sci. U. S. A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.
Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem.
Soc., 2007, 129, 8362-8379;
Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001,8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel et al.,U.S.
7,053,207, Imanishi et al., U.S.
6,268,490, Imanishi et al. U.S. 6,770,748, Imanishi et al., U.S. RE44,779;
Wengel et al., U.S. 6,794,499, Wengel et al., U.S. 6,670,461; Wengel et al., U.S. 7,034,133, Wengel et al., U.S. 8,080,644; Wengel et al., U.S. 8,034,909; Wengel et al., U.S. 8,153,365; Wengel et al., U.S. 7,572,582;
and Ramasamy et al., U.S.
6,525,191, Torsten et al., WO 2004/106356, Wengel et al., WO 1999/014226; Seth et al.,WO 2007/134181;
Seth et al., U.S. 7,547,684; Seth et al., U.S. 7,666,854; Seth et al., U.S.
8,088,746; Seth et al., U.S. 7,750,131;
Seth et al., U.S. 8,030,467; Seth et al., U.S. 8,268,980; Seth et al., U.S.
8,546,556; Seth et al., U.S. 8,530,640;
Migawa et al., U.S. 9,012,421; Seth et al., U.S. 8,501,805; Allerson et al., U52008/0039618; and Migawa et al., U52015/0191727.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA
nucleoside (described herein) may be in the a-L configuration or in the I3-D configuration.
107/Bx 09 Bx LNA (I3-D-configuration) a-L-LNA (a-L-configuration) bridge = 4'-CH2-0-2' bridge = 4'-CH2-0-2' a-L-methyleneoxy (4' -CH2-0-2') or a-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the I3-D configuration, unless otherwise specified.
In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5'-substituted and 4'-2' bridged sugars).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4' -sulfur atom and a substitution at the 2'-position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S.
7,939,677) and/or the 5' position.
In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran ("THP"). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid ("HNA"), anitol nucleic acid ("ANA"), manitol nucleic acid ("MNA") (see e.g., Leumann, CJ. Bioorg. &Med. Chem. 2002, 10, 841-854), fluoro HNA:
Fo_..õ0, ,0,=Bx P
F-HNA
("F-HNA", see e.g., Swayze et al., U.S. 8,088,904; Swayze et al., U.S.
8,440,803; and Swayze et al., U.S.
9,005,906, F-HNA can also be referred to as a F-THP or 3'-fluoro tetrahydropyran), and nucleosides comprising additional modified THP compounds having the formula:
c11 q2 T3-0¨Ko.L CI3 q6- x / Ri R2 wherein, independently, for each of said modified THP nucleoside:
Bx is a nucleobase moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the modified THP
nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3'-terminal group; qi, q2, q3, q4, q5, q6 and q7 are each, independently, H, Ci-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3, OC(=X)Ji, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is 0, S or NJi, and each Ji, 12, and 73 is, independently, H or Ci-C6 alkyl.
In certain embodiments, modified THP nucleosides are provided wherein qi, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of qi, q2, .43, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, modified THP
nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and R2 is H.
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S.
5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444;
and Summerton et al., U.S.
5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following structure:
1-0-10Bx N
I
In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are refered to herein as "modifed morpholinos."
In certain embodiments, sugar surrogates comprise acyclic moieites. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid ("PNA"), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol.
Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., U52013/130378.
Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.
2. Modified Nucleobases Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds.
In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside that does not comprise a nucleobase, referred to as an abasic nucleoside.
In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimi-dines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (CC-CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in Merigan et al., U.S. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859;
Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613;
Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S.T., Ed., CRC Press, 2008, 163-166 and 442-443.
Publications that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, Manoharan et al., US2003/0158403, Manoharan et al., U52003/0175906; Dinh et al., U.S. 4,845,205; Spielvogel et al., U.S.
5,130,302; Rogers et al., U.S. 5,134,066;
Bischofberger et al., U.S. 5,175,273; Urdea et al., U.S. 5,367,066; Benner et al., U.S. 5,432,272; Matteucci et al., U.S. 5,434,257; Gmeiner et al., U.S. 5,457,187; Cook et al., U.S.
5,459,255; Froehler et al., U.S. 5,484,908;
Matteucci et al., U.S. 5,502,177; Hawkins et al., U.S. 5,525,711; Haralambidis et al., U.S. 5,552,540; Cook et al., U.S. 5,587,469; Froehler et al., U.S. 5,594,121; Switzer et al., U.S.
5,596,091; Cook et al., U.S. 5,614,617;
Froehler et al., U.S. 5,645,985; Cook et al., U.S. 5,681,941; Cook et al., U.S. 5,811,534; Cook et al., U.S.
5,750,692; Cook et al., U.S. 5,948,903; Cook et al., U.S. 5,587,470; Cook et al., U.S. 5,457,191; Matteucci et al., U.S. 5,763,588; Froehler et al., U.S. 5,830,653; Cook et al., U.S.
5,808,027; Cook et al., U.S. 6,166,199;
and Matteucci et al., U.S. 6,005,096.
In certain embodiments, compounds targeted to a PCSK9 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
3. Modified Internucleoside Linkages The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkageIn certain embodiments, compounds described herein having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
In certain embodiments, compounds targeted to a PCSK9 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
In certain embodiments, compounds described herein comprise oligonucleotides.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond ("P=0") (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"). Representative non-phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (-CH2-N(CH3)-0-CH2-), thiodiester, thionocarbamate (-0-C(=0)(NH)-S-); siloxane (-0-SiH2-0-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
Representative chiral internucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-0-5'), amide-3 (3'-CH2-C(=0)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=0)-5'), formacetal (3'-0-CH2-0-5'), methoxypropyl, and thioformacetal (3'-S-CH2-0-5'). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S.

Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, 0, S
and CH2 component parts.
In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
In certain embodiments, oligonucleotides comprise one or more methylphosponate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosponate linkages.
In certain embodiments, one methylphosponate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.
In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased.
In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.
4. Certain Motifs In certain embodiments, compounds described herein comprise oligonucleotides.
Oligonucleotides can have a motif, e.g. a pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).
a. Certain Sugar Motifs In certain embodiments, compounds described herein comprise oligonucleotides.
In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include but are not limited to any of the sugar modifications discussed herein.
In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or "wings" and a central or internal region or "gap." The three regions of a gapmer motif (the 5' -wing, the gap, and the 3' -wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3' -most nucleoside of the 5' -wing and the 5' -most nucleoside of the 3' -wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5'-wing differs from the sugar motif of the 3'-wing (asymmetric gapmer).
In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides.
In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiment, each nucleoside of the gap of a gapmer is an unmodified 2' -deoxy nucleoside.
In certain embodiments, the gapmer is a deoxy gapmer. In such embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2' -deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2' -deoxy nucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.
In certain embodiments, a modified oligonucleotide has a fully modified sugar motif wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety.
In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif wherein each nucleoside of the region comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2' -modification.
In certain embodiments, a modified oligonucleotide can comprise a sugar motif described in Swayze et al., US2010/0197762; Freier et al., US2014/0107330; Freier et al., US2015/0184153; and Seth et al., US2015/0267195, each of which is incorporated by reference in its entirety herein.
b. Certain Nucleobase Motifs In certain embodiments, compounds described herein comprise oligonucleotides.
In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methylcytosines.
In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3' -end of the oligonucleotide.
In certain embodiments the block is within 3 nucleosides of the 3' -end of the oligonucleotide. In certain embodiments, the block is at the 5' -end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5' -end of the oligonucleotide.
In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2' -deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.
c. Certain Internucleoside Linkage Motifs In certain embodiments, compounds described herein comprise oligonucleotides.
In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linking group is a phosphate internucleoside linkage (P=0). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate (P=S). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages.
In certain embodiments, the terminal internucleoside linkages are modified.
5. Certain Modified Oligonucleotides In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification, motifs, and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Furthermore, in certain instances, an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a regions of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A
consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B
consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20). Herein, if a description of an oligonucleotide is silent with respect to one or more parameter, such parameter is not limited. Thus, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of nucleobase sequence.
Certain Conjugated Compounds In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3' and/or 5' -end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3' -end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3' -end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5' -end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5' -end of oligonucleotides.
In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a compound has a nucleobase sequence that is complementary to a target nucleic acid. In certain embodiments, oligonucleotides are complementary to a messenger RNA (mRNA). In certain embodiments, oligonucleotides are complementary to a sense transcript.
Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearanceIn certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
Certain conjugate groups and conjugate moieties have been described previously, for example:
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem.
Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;
Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
Ther., 1996, i, 923-937),_a tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038/mtna.2014.72 and Nishina et al., Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., W02014/179620).
1. Conjugate Moieties Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
2. Conjugate linkers Conjugate moieties are attached to oligonucleotides through conjugate linkers.
In certain compounds, a conjugate group is a single chemical bond (i.e. conjugate moiety is attached to an oligonucleotide via a conjugate linker through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.
In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
Examples of conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include but are not limited to substituted or unsubstituted Ci-Cio alkyl, substituted or unsubstituted C2-Cio alkenyl or substituted or unsubstituted C2-Cio alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoy1-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the compound through cleavable bonds.
In certain embodiments, such cleavable bonds are phosphodiester bonds.

Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which a compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, a compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is more than 30.
Alternatively, an compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound is no more than 30. Unless otherwise indicated conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide.
For example, in certain circumstances compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugate may comprise one or more cleavable moieties, typically within the conjugate linker. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.
In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides.
In certain such embodiments, one or more linker-nucleosides are linked to one another and/or to the remainder of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2'-deoxy nucleoside that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2'-deoxyadenosine.
3. Certain Cell-Targeting Conjugate Moieties In certain embodiments, a conjugate group comprises a cell-targeting conjugate moiety. In certain embodiments, a conjugate group has the general formula:
ILigand¨Tetherl¨n [Branching group I¨ [Conjugate Linker [.¨[Cleavable Moiety I¨k1 m J
Cell-targeting moiety wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or O.
In certain embodiments, n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is 0 and k is 1. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1.
In certain embodiments, n is 3, j is 1 and k is 1.
In certain embodiments, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.
In certain embodiments, the cell-targeting moiety comprises a branching group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system.
In certain embodiments, each tether of a cell-targeting moiety comprises one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amid, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group. In certain embodiments, each tether comprises a chain from about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain from about 10 to about 18 atoms in length. In certain embodiments, each tether comprises about 10 atoms in chain length.
In certain embodiments, each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, each ligand has an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine (GalNAc), mannose, glucose, glucoseamine and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the cell-targeting moiety comprises 3 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 2 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 1 GalNAc ligand.
In certain embodiments, each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J. Med. Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety).
In certain such embodiments, each ligand is an amino sugar or a thio sugar.
For example, amino sugars may be selected from any number of compounds known in the art, such as sialic acid, a-D-galactosamine, I3-muramic acid, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4 -formamido-2,3 -di-O-methyl-D -mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-glycoloyl-a-neuraminic acid. For example, thio sugars may be selected from 5-Thio-I3-D-glucopyranose, methyl 2,3,4-tri-0-acety1-1-thio-6-0-trityl-a-D-glucopyranoside, 4-thio-3-D-galactopyranose, and ethyl 3,4,6,7-tetra-0-acety1-2-deoxy-1,5-dithio-a-D-g/uco-heptopyranoside.

In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:
HO OH
H
HO---T2--\V NyH2o AcHN 0 \
HO OH
HO 0.,(1.,, NI.re(y N¨

AcHN 0 HO OH
H
HO---T2--\V0)¨)zVN^/
AcHN 0 In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:
HO OH
H
4 II \
AcHN 0 HO OH
4 H s ________________________________________ N
AcHN 0 HO OH

AcHN 0 =
In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:

H21\11r0 AcHN 0 \

HO 2 NMNIreCY ____________ N

AcHN 0 AcHN 0 In certain embodiments, compounds described herein comprise a conjugate group described herein as "LICA-1". LICA-1 is shown below without the optional cleavable moiety at the end of the conjugate linker:

HO OH
H
AcHN 0 \

......72..\7,-, H

AcHN 0 HO OH
H
AcHN 0 In certain embodiments, compounds described herein comprise LICA-1 and a cleavable moiety within the conjugate linker have the formula:
Oligo I
Ligand Cleavable moiety I oil 1 - -Tether H0¨P=0 HO OH -H
HO---112-\7 N1rio ¨ ¨ 0 ¨
AcHN \ ( __ _ 0 NH

H H
1)3 HO--i2-\, 4 Ny(¨)0 _____________________________ N _____ \
AcHN 0 ¨ 0 ¨
Conjugate HO OH linker H
HO---112-\7 4 /
AcHN 0 _ _ Branching group Cell targeting moiety .
wherein oligo is an oligonucleotide.
Representative publications that teach the preparation of certain of the above noted conjugate groups and compounds comprising conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, US
5,994,517, US 6,300,319, US 6,660,720, US 6,906,182, US 7,262,177, US 7,491,805, US 8,106,022, US 7,723,509, US
9,127,276, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, Biessen et al., J. Med.
Chem. 1995, 38, 1846-1852, Lee et al., Bioorganic & Medicinal Chemistry 2011,19, 2494-2500, Rensen et al., J. Biol. Chem. 2001, 276, 37577-37584, Rensen et al., J. Med. Chem. 2004, 47, 5798-5808, Sliedregt et al., J. Med. Chem. 1999, 42, 609-618, and Valentijn et al., Tetrahedron, 1997, 53, 759-770, each of which is incorporated by reference herein in its entirety.
In certain embodiments, compounds described herein comprise modified oligonucleotides comprising a gapmer or fully modified motif and a conjugate group comprising at least one, two, or three GalNAc ligands.
In certain embodiments compounds described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et at, Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J
Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8,762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43;
Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135;
Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46;
Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J
Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808;
Rensen et al., Arterioscler Thromb Vase Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571;
Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications W01998/013381; W02011/038356; W01997/046098; W02008/098788;
W02004/101619;
W02012/037254; W02011/120053; W02011/100131; W02011/163121; W02012/177947;
W02013/033230; W02013/075035; W02012/083185; W02012/083046; W02009/082607;
W02009/134487; W02010/144740; W02010/148013; W01997/020563; W02010/088537;
W02002/043771; W02010/129709; W02012/068187; W02009/126933; W02004/024757;
W02010/054406; W02012/089352; W02012/089602; W02013/166121; W02013/165816;
U.S. Patents 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022;
7,491,805; 7,491,805;
7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548;
8,344,125; 8,313,772;
8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916;
8,435,491; 8,404,862;
7,851,615; Published U.S. Patent Application Publications U52011/0097264;
U52011/0097265;

US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044;
US2010/0240730;
US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;
US2009/0286973;
US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041;
US2009/0203135;
US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760;
US2012/0157509;
US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512;
US2013/0236968;
US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.
Compositions and Methods for Formulating Pharmaceutical Compositions Compounds described herein may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, or extent of disease.
Certain embodiments provide pharmaceutical compositions comprising one or more compounds or a salt thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compounds comprise or consist of a modified oligonucleotide. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more compound and sterile PBS.
In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, or extent of disease.
A compound described herein targeted to PCSK9 nucleic acid can be utilized in pharmaceutical compositions by combining the compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection.
Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising a compound targeted to PCSK9 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the compound comprises or consists of a modified oligonucleotide provided herein.

Pharmaceutical compositions comprising compounds provided herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of a compound which are cleaved by endogenous nucleases within the body, to form the active compound.
In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.
Certain Selected Compounds Approximately 1540 newly designed compounds and a few previously disclosed compounds of various lengths, chemistries, and motifs were tested for their effect on human PCSK9 mRNA in vitro in several cell types (Example 1-2). Of 1540 compounds tested for potency at a single dose in vitro, 183 selected compounds were tested for dose dependent inhibition in HepG2 cells (Example 2). Of the 183 compounds tested by dose response assays, 134 oligonucleotides were selected for single dose in vivo tolerability in rodents. These 134 oligonucleotides were conjugated at the 3' -end with THA-C6-GalNAc3-(3R,55)-5-(hydroxymethyl) pyrrolidin-3-ol phosphate endcap (henceforth referred to as 3' -THA).
In the single dose rodent tolerability model , BALB/c mice are dosed with a high concentration of 3' -THA oligonucleotide and liver function markers, such as alanine transaminase and aspartate transaminase, are measured (Example 3). ISIS 863413 (SEQ ID NO: 1096), ISIS 863419 (SEQ ID NO:
1419), ISIS 863424 (SEQ
ID NO: 1297), ISIS 863425 (SEQ ID NO: 1528), ISIS 863427 (SEQ ID NO: 1145), ISIS 863433 (SEQ ID NO:
1223), ISIS 863434 (SEQ ID NO: 377), ISIS 863436 (SEQ ID NO: 763), ISIS 863437 (SEQ ID NO: 994), ISIS
863438 (SEQ ID NO: 1071), ISIS 863439 (SEQ ID NO: 1147), ISIS 863441 (SEQ ID
NO: 1302), ISIS 863444 (SEQ ID NO: 1149), ISIS 863445 (SEQ ID NO: 455), ISIS 863448 (SEQ ID NO:
1380), ISIS 863452 (SEQ
ID NO: 691), ISIS 863472 (SEQ ID NO: 926), ISIS 863473 (SEQ ID NO: 1233), ISIS
863474 (SEQ ID NO:
619), ISIS 863475 (SEQ ID NO: 388), ISIS 863477 (SEQ ID NO: 1542), ISIS 863479 (SEQ ID NO: 1158), ISIS 863480 (SEQ ID NO: 1235), ISIS 863481 (SEQ ID NO: 389), ISIS 863482 (SEQ
ID NO: 1312), ISIS
863483 (SEQ ID NO: 1240), ISIS 863484 (SEQ ID NO: 549), ISIS 863485 (SEQ ID
NO: 550), ISIS 863486 (SEQ ID NO: 781), ISIS 863489 (SEQ ID NO: 939), ISIS 863490 (SEQ ID NO: 1016), ISIS 863491 (SEQ ID
NO: 1243), ISIS 863493 (SEQ ID NO: 629), ISIS 863494 (SEQ ID NO: 1017), ISIS
863495 (SEQ ID NO:
1092), ISIS 863496 (SEQ ID NO: 1244), ISIS 863497 (SEQ ID NO: 554), ISIS
863498 (SEQ ID NO: 478), ISIS 863499 (SEQ ID NO: 1094), ISIS 863502 (SEQ ID NO: 563), ISIS 863506 (SEQ
ID NO: 410), ISIS

863507 (SEQ ID NO: 1411), ISIS 863509 (SEQ ID NO: 721), ISIS 863510 (SEQ ID
NO: 1258), ISIS 863511 (SEQ ID NO: 645), ISIS 863512 (SEQ ID NO: 955), ISIS 863514 (SEQ ID NO: 1413), ISIS 863516 (SEQ ID
NO: 881), ISIS 863517 (SEQ ID NO: 648), ISIS 863518 (SEQ ID NO: 725), ISIS
863520 (SEQ ID NO: 1111), ISIS 863522 (SEQ ID NO: 1188), ISIS 863524 (SEQ ID NO: 885), ISIS 863525 (SEQ
ID NO: 504), ISIS
863526 (SEQ ID NO: 1043), ISIS 863527 (SEQ ID NO: 1195), ISIS 863531 (SEQ ID
NO: 426), ISIS 863533 (SEQ ID NO: 427), ISIS 863536 (SEQ ID NO: 585), ISIS 863537 (SEQ ID NO: 1047), ISIS 863538 (SEQ ID
NO: 353), ISIS 863539 (SEQ ID NO: 1352), ISIS 863541 (SEQ ID NO: 1203), ISIS
863545 (SEQ ID NO:
1516), ISIS 863547 (SEQ ID NO: 1213), ISIS 863548 (SEQ ID NO: 367), ISIS
863549 (SEQ ID NO: 1061), ISIS 863550 (SEQ ID NO: 523), ISIS 863552 (SEQ ID NO: 831), and ISIS 863553 (SEQ ID NO: 908) were considered tolerable in this study and were selected for further evaluation in a transgenic mouse model.
In the PCSK9 transgenic mice tolerability model, mice were dosed with 3' -THA
conjugated oligonucleotides and plasma levels of alanine transaminase, aspartate transaminase, cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides were measured (Example 4). ISIS
863433, ISIS 863490, ISIS
863512, ISIS 863527, ISIS 863538, ISIS 863425, ISIS 863438, ISIS 863439, ISIS
863444, ISIS 863413, ISIS
863434, ISIS 863436, ISIS 863494, and ISIS 863539 were found tolerable. The selected oligonucleotide sequences were conjugated at the 5' -end with Trishexylamino-(THA)-C6GalNAC3 endcap (henceforth referred to as 5' -THA) and tested in the CD-1 mouse model. The 5'THA
conjugated oligonucleotides are ISIS
845219, ISIS 863568, ISIS 863576, ISIS 863577, ISIS 863579, ISIS 863581, ISIS
863582, ISIS 863587, ISIS
863633, ISIS 863637, ISIS 863655, ISIS 863670, ISIS 863681, and ISIS 863682.
In the CD-1 mice tolerability model, mice were dosed for six weeks with varying doses of antisense oligonucleotide and tolerability markers, such as liver and kidney function markers (plasma levels of ALT, AST, albumin, BUN, creatinine, and bilirubin), hematology markers (blood cell counts for RBC, WBC, platelets, neutrophils, lymphocytes, and monocytes, as well as hemoglobin, hematocrit, and MCV content), and body and organ weights were measured (Example 5). Only those oligonucleotides were selected for further .. evaluation in the rat model, where treatment yielded plasma levels of such markers which were within the expected range after treatment with antisense olionucleotides. ISIS 863568, ISIS 863576, ISIS 863579, ISIS
863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 were selected for further study in the rat model.
In the Sprague-Dawley rat model, similarly, the rats were dosed for 6 weeks with antisense oligonucleotides and tolerability markers, such as liver function markers (plasma levels of ALT, AST, albumin, BUN, creatinine, and bilirubin), kidney function markers (urine levels of creatinine and total protein), and weekly body weights, and final organ weights were measured (Example 6).
These oligonucleotides were also tested for their viscosity (Example 8) and all found to be optimal in their viscosity under the criteria tested.

ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS
863670, and ISIS 863681 were tested in a dose-dependent four-week study in the PCSK9 transgenic mice model for efficacy (Example 7). At the end of 4 weeks, mice liver was analyzed for PCSK9 mRNA expression levels and the ED50 was calculated. In addition, PCSK9 plasma protein levels, LDL-cholesterol plasma levels, and LDL-receptor levels in the liver were measured. It was observed that treatment with the antisense oligonucleotides resulted in inhibition of PCSK9 liver mRNA expression levels, reduction in LDL-cholesterol levels in the plasma, and corresponding increase in LDL receptor levels in the liver. Specifically, ISIS 863633 treatment was found to be efficacious.
ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS
863670, and ISIS 863681 were tested for activity, pharmacokinetic profile and tolerability in a 12-week study in cynomolgus monkeys (Example 9). Treatment with the compounds that were fully cross-reactive with the rhesus monkey gene sequence caused reduction of PCSK9 mRNA expression in liver tissue. Specifically, treatment with ISIS 863633, ISIS 863670, and ISIS 863681 caused a reduction of PCSK9 mRNA expression in liver tissue, compared to the PBS control. It was noted that ISIS 863633 caused the highest reduction of PCSK9 mRNA expression compared to the PBS control. Changes in PCSK9 protein levels, LDL-cholesterol levels, total cholesterol levels, and hepatic LDL-receptor levels are a consequence of inhibition of PCSK9 mRNA levels. Treatment with ISIS 863633, ISIS 863670, and ISIS 863681 caused decreases in PCSK9 protein levels, LDL-cholesterol levels, total cholesterol levels, and induction of hepatic LDL-receptor levels, with ISIS
863633 causing the highest change. Treatment with the antisense oligonucleotides did not cause any changes in HDL-cholesterol or triglyceride levels. Hence, in terms of activity, ISIS
863633 was the most effective in the monkey study. Treatment with the compounds was well tolerated in the monkeys, in particular, treatment with ISIS 863633.
The new compounds were compared with previously designed compounds, including ISIS 405879 and ISIS 405995, which have previously been determined to be some of the most potent antisense compounds in vitro (see e.g., U.S. Patent 8,084,437), as well as ISIS 431131 and ISIS
480604, which have been previously described in U.S. Patent 9,127,276 (Example 10). The head-to-head comparison demonstrated that ISIS
863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS
863655, ISIS 863670, and ISIS 863681 were more efficacious than any of the previously publicly disclosed oligonucleotides.
Accordingly, provided herein are compounds with any one or more of the improved properties. In certain embodiments, the compounds as described herein are potent and tolerable.
EXAMPLES
The Examples below describe the screening process to identify lead compounds targeted to PCSK9.
Out of over 1540 oligonucleotides that were screened, ISIS 863568, ISIS
863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 emerged as the top lead compounds.

In particular, ISIS 863633 exhibited the best combination of properties in terms of potency and tolerability out of over 1540 oligonucleotides.
Non-limiting disclosure and incorporation by reference Although the sequence listing accompanying this filing identifies each sequence as either "RNA" or "DNA" as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as "RNA" or "DNA" to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2' -OH sugar moiety and a thymine base could be described as a DNA
having a modified sugar (2' -OH for the natural 2'-H of DNA) or as an RNA
having a modified base (thymine (methylated uracil) for natural uracil of RNA). As provided herein, a designation of '0' for mRNA inhibition assays merely indicates that the antisense oligonucleotide did not inhibit mRNA expression levels.
Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence "ATCGATCG" encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence "AUCGAUCG" and those having some DNA bases and some RNA bases such as "AUCGATCG" and compounds having other modified nucleobases, such as "ATmCGAUCG," wherein mC indicates a cytosine base comprising a methyl group at the 5-position.
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1: Antisense inhibition of human PCSK9 in HepG2 cells by 3-10-3 cEt gapmers Antisense oligonucleotides were designed targeting a PCSK9 nucleic acid and were tested for their effects on PCSK9 mRNA in vitro. The chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers. The gapmers are 16 nucleosides in length, wherein the central gap segment is comprised of ten 2' -deoxynucleosides and is flanked by wing segments on the 5' end and the 3' end of three nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a cEt sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
"Start site" indicates the 5' -most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3' -most nucleoside to which the gapmer is targeted human gene sequence.

Each gapmer listed in the Tables below is targeted to either the human PCSK9 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_174936.3) or the human PCSK9 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NC_000001.11 truncated from nucleotides 55036001 to 55068000). `n/a' indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below.
Study 1 Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. ISIS 431131, previously disclosed in W02014179620, was also included in the study as a benchmark oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR.
Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA levels.
PCSK9 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN .
Results are presented as percent inhibition of PCSK9, relative to untreated control cells. Several of the newly designed oligonucleotides were more potent than the previously disclosed oligonucleotide, ISIS 431131.
Table 1 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2: SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 2 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID
2: SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 3 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ
SEQ
ID: 1 ID: 1 % ID: 2 ID
2: SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 4 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID
2: SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Study 2 Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 3,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA
levels. PCSK9 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN . Results are presented as percent inhibition of PCSK9, relative to untreated control cells.

Table 5 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 6 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ
SEQ
ID: 1 ID: 1 PCSK9 (% ID: 2 ID 2: SEQ
ISIS No Sequence Start Stop inhibition) Start Stop ID NO
Site Site Site Site Study 3 Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA
levels. PCSK9 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN . Results are presented as percent inhibition of PCSK9, relative to untreated control cells.
Table 7 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 8 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 9 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 10 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 11 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 12 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2: SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 13 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 14 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 15 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 16 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 17 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 18 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 19 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Table 20 Inhibition of PCSK9 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2 SEQ SEQ SEQ SEQ
ID: 1 ID: 1 % ID: 2 ID 2:
SEQ
ISIS No Sequence Start Stop inhibition Start Stop ID NO
Site Site Site Site Example 2: Dose-dependent antisense inhibition of human PCSK9 in HepG2 cells by 3-10-3 cEt gapmers Select gapmers from Example 1 exhibiting in vitro inhibition of PCSK9 mRNA
were tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below.
Study 1 Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 46.88 nM, 187.5 nM, 750 nM, and 3,000 nM concentrations of antisense oligonucleotide, as specified in the Tables below. ISIS 405879, previously disclosed in W02008066776 was also included in the study as a benchmark oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA levels. PCSK9 mRNA
levels were adjusted according to total RNA content, as measured by RIBOGREEN . Results are presented as percent inhibition of PCSK9, relative to untreated control cells.

The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented. PCSK9 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Several of the newly designed antisense oligonucleotides demonstrated superior efficacy compared to the previously disclosed oligonucleotide, ISIS 405879. Specifically, ISIS 848542, ISIS
848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040, ISIS 849171, and ISIS 849236 demonstrated superior efficacy compared to the previously disclosed oligonucleotide, ISIS 405879.
Table 21 46.875 187.5 750 3,000 ICso ISIS No nM nM nM nM ( M) 405879 13 6 3 29 >3.00 466847 1 19 65 94 0.47 468497 12 27 60 83 0.48 848219 0 0 54 88 0.91 848241 0 5 31 63 1.78 848283 18 27 54 85 0.48 848306 0 19 53 91 0.62 848323 0 0 0 50 >3.00 848376 0 3 12 43 >3.00 848430 0 30 52 81 0.62 848431 67 79 92 96 <0.05 848500 6 30 55 82 0.54 848539 0 0 0 55 >3.00 848542 10 30 66 86 0.41 848560 12 32 76 91 0.33 848567 0 1 42 89 0.85 848570 23 27 67 87 0.35 848572 18 44 71 81 0.30 848574 0 5 68 95 0.55 848575 0 17 51 80 0.75 848576 11 18 45 74 0.86 848583 3 18 62 88 0.52 848584 0 22 54 89 0.57 848585 1 22 67 92 0.45 848593 0 25 53 83 0.63 848596 18 40 76 93 0.26 848597 22 42 78 87 0.24 848598 0 18 62 95 0.53 848616 0 14 55 88 0.66 848618 0 0 56 77 0.96 848619 3 7 41 74 1.07 848629 21 34 78 78 0.31 848630 7 22 65 83 0.50 848632 25 34 68 89 0.29 848635 0 0 49 83 0.92 848636 5 34 77 76 0.42 848649 5 10 43 78 0.89 848651 4 0 48 88 0.88 848652 0 6 28 80 1.16 848656 1 13 52 80 0.73 848670 0 38 84 85 0.39 848671 26 28 70 81 0.34 848672 0 11 27 73 1.37 848674 36 31 69 89 0.24 848677 2 32 69 90 0.41 848679 0 11 55 81 0.73 848680 11 20 64 81 0.52 848685 0 14 33 62 1.63 848687 0 25 63 90 0.51 848688 0 23 61 89 0.55 848690 15 21 78 84 0.39 848692 31 45 80 97 0.17 848693 8 23 69 88 0.44 848694 27 43 75 93 0.21 848701 0 10 39 77 1.02 848704 11 5 43 78 0.94 848725 0 9 51 81 0.79 848729 26 33 63 92 0.30 848731 0 15 60 89 0.55 848732 12 71 67 90 0.21 848736 6 18 40 73 1.00 848740 2 18 59 86 0.57 848748 9 42 60 85 0.39 848756 3 1 55 85 0.72 848759 18 37 75 94 0.28 848761 20 35 68 81 0.35 848764 0 8 37 64 1.52 848770 0 4 63 90 0.63 848771 3 23 57 93 0.50 848772 15 32 66 82 0.40 848774 0 8 64 90 0.61 848790 0 0 33 73 1.49 848795 9 42 62 91 0.35 848811 7 5 46 86 0.75 848814 17 20 64 89 0.43 848826 7 57 77 93 0.24 848830 0 35 45 86 0.62 848832 0 40 71 78 0.52 848833 19 43 68 81 0.32 848838 0 36 72 89 0.39 848841 5 24 62 86 0.50 848844 0 22 61 89 0.53 848849 9 48 79 92 0.26 848850 0 38 68 97 0.43 848854 2 7 58 84 0.66 848875 3 17 58 79 0.65 848890 0 6 27 65 1.74 848898 7 14 79 87 0.43 848927 12 25 75 95 0.35 848928 0 0 45 78 1.01 848971 0 21 54 82 0.65 848999 6 29 34 64 1.38 849007 0 4 41 78 1.02 849008 0 39 74 90 0.42 849013 4 17 47 86 0.67 849016 0 23 56 79 0.65 849020 0 13 40 67 1.28 849021 10 34 65 80 0.44 849030 2 28 58 86 0.52 849036 2 1 37 76 1.16 849040 28 55 79 96 0.15 849044 26 39 72 91 0.28 849048 0 30 60 79 0.61 849069 12 12 40 74 1.02 849071 2 31 65 92 0.42 849084 0 0 0 43 >3.00 849085 20 39 64 89 0.31 849086 13 40 69 81 0.36 849106 20 24 55 80 0.53 849120 0 15 61 80 0.64 849123 9 48 82 84 0.27 849133 0 0 29 70 1.62 849135 25 54 79 95 0.17 849162 0 4 35 75 1.19 849169 34 63 72 83 0.12 849171 9 60 63 83 0.30 849177 0 16 69 75 0.68 849178 0 22 53 83 0.65 849181 6 8 39 84 0.86 849189 24 44 81 90 0.21 849200 0 0 34 70 1.48 849205 7 29 51 88 0.52 849216 0 15 28 66 1.66 849225 23 47 78 94 0.21 849227 7 35 74 94 0.34 849233 9 42 68 93 0.32 849236 0 1 57 88 0.73 849239 18 38 74 86 0.30 849266 0 22 66 86 0.51 849267 18 69 72 94 0.17 849285 3 18 45 84 0.71 849288 0 0 0 52 >3.00 849303 18 33 57 72 0.54 849305 25 28 62 82 0.40 849309 10 18 59 82 0.57 849331 0 10 65 93 0.57 849332 0 0 48 76 1.09 849345 0 0 62 80 0.78 849354 0 0 43 64 1.49 849357 5 21 45 75 0.84 849358 0 10 45 79 0.88 849359 3 22 46 88 0.62 849372 0 15 29 82 1.04 849383 2 0 33 71 1.43 849386 11 18 51 84 0.60 Study 2 Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 78.13 nM, 312.5 nM, 1,250 nM, and 5,000 nM concentrations of antisense oligonucleotide, as specified in the Tables below. ISIS 431131, previously disclosed in W02014179620, was also included in the study as a benchmark oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA levels. PCSK9 mRNA
levels were adjusted according to total RNA content, as measured by RIBOGREEN . Results are presented as percent inhibition of PCSK9, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented. PCSK9 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Several of the newly designed antisense oligonucleotides demonstrated superior efficacy compared to the previously disclosed oligonucleotide, ISIS 431131.
Table 22 78.125 312.5 1,250 5,000 IC50 ISIS No nM nM nM nM ( M) 431131 18 16 30 55 >5 849169 23 57 86 92 0.28 859341 24 48 69 85 1.54 859346 27 54 80 94 0.27 859358 0 30 69 89 0.76 859361 28 53 73 94 0.30 859363 38 50 79 82 0.20 859384 19 59 76 94 0.30 859387 36 66 79 96 0.16 859392 14 33 63 81 0.96 859393 20 59 90 96 0.27 859394 43 65 80 96 0.12 859395 37 51 82 91 0.21 859401 27 64 88 94 0.20 859408 0 59 61 92 0.55 859419 22 58 77 84 0.31 859459 33 55 91 93 0.19 859462 31 56 78 96 0.24 859469 26 48 76 83 0.36 859472 19 35 67 87 0.56 859504 19 47 59 80 0.57 859529 19 50 88 94 0.32 859531 33 52 76 69 0.29 859532 24 55 79 90 0.30 859533 9 40 77 91 0.51 859534 25 49 85 85 0.70 859551 25 41 51 78 0.69 859552 0 17 53 78 1.36 859553 24 45 79 94 0.35 859561 28 54 71 92 0.30 859571 35 34 76 73 0.43 859584 19 51 85 95 0.32 859598 33 55 73 83 0.25 859601 23 39 63 90 0.50 859603 0 6 70 71 1.25 859605 9 42 67 83 0.62 859611 21 22 47 76 1.16 Example 3: Tolerability of 3'-conjugated 3-10-3 cEt gapmers targeting human PCSK9 in BALB/c mice ISIS oligonucleotides selected from the studies above were conjugated with 3' -THA-C6-GalNAc3-(3R,5S)-5-(hydroxymethyl) pyrrolidin-3-ol phosphate endcap (henceforth referred to as 3' -THA). The hundred and sixty two 3'-THA-conjugated ISIS antisense oligonucleotides evaluated for changes in the levels of plasma chemistry markers are presented in the Table below. 'Parent Oligo' indicates the ISIS
oligonucleotide that has been described in the studies above and that was conjugated with 3'-THA and tested in this study.
Table 23 3'-conjugated 3-10-3 cEt gapmers selected for tolerability evaluation in BALB/c mice ISIS No Parent SEQ ID
Oligo NO

BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were administered a single dose of oligonucleotide. Plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). ISIS
oligonucleotides that caused changes in the levels of ALT and/or AST outside the expected range for antisense oligonucleotides were excluded in further studies. ISIS 863413, ISIS
863419, ISIS 863424, ISIS
863425, ISIS 863427, ISIS 863433, ISIS 863434, ISIS 863436, ISIS 863437, ISIS
863438, ISIS 863439, ISIS
863441, ISIS 863444, ISIS 863445, ISIS 863448, ISIS 863452, ISIS 863472, ISIS
863473, ISIS 863474, ISIS
863475, ISIS 863477, ISIS 863479, ISIS 863480, ISIS 863481, ISIS 863482, ISIS
863483, ISIS 863484, ISIS
863485, ISIS 863486, ISIS 863489, ISIS 863490, ISIS 863491, ISIS 863493, ISIS
863494, ISIS 863495, ISIS
863496, ISIS 863497, ISIS 863498, ISIS 863499, ISIS 863502, ISIS 863506, ISIS
863507, ISIS 863509, ISIS
863510, ISIS 863511, ISIS 863512, ISIS 863514, ISIS 863516, ISIS 863517, ISIS
863518, ISIS 863520, ISIS
863522, ISIS 863524, ISIS 863525, ISIS 863526, ISIS 863527, ISIS 863531, ISIS
863533, ISIS 863536, ISIS
863537, ISIS 863538, ISIS 863539, ISIS 863541, ISIS 863545, ISIS 863547, ISIS
863548, ISIS 863549, ISIS
863550, ISIS 863552, and ISIS 863553 were considered tolerable in this study and were selected for further evaluation.
Example 4: Effect of antisense inhibition of PCSK9 in transgenic mouse model A transgenic mouse model was developed at UCI using the human PCSK9 genomic construct, which was contained in fosmid ABC7-611722G24 and restricted with Nhel to produce a DNA fragment containing the entire genomic sequence as well as 8 Kb of 5' and 0.4 Kb of 3' noncoding sequence. PCSK9 transgenic mice were produced by random insertion via micronucleus injection. Progeny expressed human PCSK9 mRNA in the liver and secreted human PCSK9 plasma protein.
The efficacy of 3'-THA-conjugated ISIS oligonucleotides was evaluated in this model (referred to herein as Tg mice). The ISIS oligonucleotides tested are presented in the Table below. 'Parent ISIS No' indicates the ISIS oligonucleotide that has been described in the studies above and that was conjugated with 3' -THA and tested in this study.

Table 24 ISIS oligonucleotides tested in Tg mice Parent SEQ ID
ISIS No Sequence ISIS No NO

Treatment PCSK9 transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (AS0s) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The PCSK9 transgenic mice received subcutaneous injections of ISIS
oligonucleotide at a dose of 2.5 mg/kg twice per week for 2 weeks. Two groups of mice received subcutaneous injections of PBS for 2 weeks. The saline-injected groups served as the control group to which oligonucleotide-treated groups were compared.

Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function of these mice, plasma levels of transaminases (ALT and AST), cholesterol (CHOL), HDL cholesterol (HDL), LDL cholesterol (LDL), and triglycerides (TRIG) were measured on day 12 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the Table below. ISIS
oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.
Table 25 Plasma chemistry markers in Tg mice plasma ALT AST CHOL HDL LDL TRIG
Treatment (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) Example 5: Tolerability of ISIS oligonucleotides targeting human PCSK9 in CD!
mice ISIS oligonucleotides selected from the studies above were conjugated with 5'-Trishexylamino-(THA)-C6GalNAC3 endcap (henceforth referred to as 5'-THA). CD1 mice (Charles River, MA) are a multipurpose mice model, frequently utilized for safety and efficacy testing.
The mice were treated with ISIS
antisense oligonucleotides, selected from studies described above and conjugated with 5'-THA, and evaluated for changes in the levels of various plasma chemistry markers.
Study 1 Treatment Groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with 15 mg/kg of ISIS oligonucleotides. One group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
The ISIS oligonucleotides tested are presented in the Table below.
'Unconjugated parent ISIS No.' refers to the ISIS oligonucleotide described in the in vitro studies above with the same sequence. '3'-THA
counterpart ISIS No.' refers to the 3'THA conjugated oligonucleotide with the same sequence and evaluated in the transgenic mice study above.
Table 26 ISIS oligonucleotides tested in CD1 mice tolerability study Unconjugated 3'-THA SEQ
ISIS No. parent ISIS counterpart Sequence ID
No. ISIS No NO

Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the Table below. Treatment with the ISIS oligonucleotides did not cause any changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides.
Table 27 Plasma chemistry markers in CD1 mice plasma at Day 45 ALT AST ALB BUN CRE TBIL
(IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 25 41 2.6 20.1 0.07 0.13 ISIS 863576 125 134 2.8 24.5 0.10 0.14 ISIS 863633 35 60 2.6 21.8 0.10 0.14 ISIS 863655 77 84 2.4 20.0 0.06 0.13 ISIS 863670 25 61 2.7 22.5 0.09 0.15 ISIS 863681 27 47 2.8 26.5 0.12 0.14 Hematology assays Blood obtained from all mice groups were analyzed for RBC, WBC, platelets, neutrophils, lymphocytes, and monocyte counts, as well as hemoglobin, HCT and MCV levels.
The results are presented in the Table below. Treatment with the ISIS oligonucleotides did not cause any changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides.
Table 28 Blood cell count in CD1 mice at Day 45 RBC Platelets WBC Neutrophils Lymphocytes Monocytes (x 106/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) PBS 9.0 1288 11.3 12.6 9128 515 863576 8.3 1251 9.4 18.8 6643 316 863633 9.4 1338 10.1 13.5 8009 698 863655 8.9 1320 8.0 10.0 5919 473 863670 9.7 1335 8.3 21.3 6514 402 863681 9.5 1329 7.6 11.0 6376 310 Table 29 Hematology markers in CD1 mice at Day 45 Hemoglobin HCT
MCV (fL) (g/dL) (0/0) PBS 14.0 43 48 863576 12.9 40 48 863633 14.6 45 49 863655 14.1 44 50 863670 14.7 44 46 863681 14.6 45 47 Body and organ weight measurements To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured every week and are presented in the Table below.
Organ weights were measured and the data is also presented in the Table below.
The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.

Table 30 Body weights (g) in CD1 mice Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 (day 1) (day 8) (day 15) (day 22) (day 29) (day 36) (day 43) Table 31 Organ weights (g) in CD1 mice Liver Spleen Kidney PBS 2.25 0.16 0.63 863576 2.32 0.19 0.59 863633 2.25 0.11 0.50 863655 2.55 0.15 0.61 863670 1.89 0.11 0.51 863681 2.25 0.10 0.47 Study 2 Treatment Groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with 15 mg/kg of ISIS oligonucleotides. One group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
The ISIS oligonucleotides tested are presented in the Table below.
'Unconjugated parent ISIS No.' refers to the ISIS oligonucleotide described in the in vitro studies above with the same sequence. '3'-THA
counterpart ISIS No.' refers to the 3'THA conjugated oligonucleotide with the same sequence and described in the transgenic mice study above.
Table 32 ISIS oligonucleotides tested in CD1 mice tolerability study 3'-THA
Unconjugated ISIS No. counterpart Sequence SEQ ID NO
parent ISIS No.
ISIS No Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).
The results are presented in the Table below. Treatment with the ISIS
oligonucleotides did not cause any changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides.
Table 33 Plasma chemistry markers in CD1 mice plasma at Day 45 ALT AST ALB BUN CRE TBIL
(IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 25 51 2.6 25.8 0.10 0.12 ISIS 863568 72 67 2.6 20.4 0.10 0.14 ISIS 863581 74 77 2.7 25.0 0.10 0.14 ISIS 863582 71 89 2.8 24.4 0.11 0.13 ISIS 863587 60 60 2.5 24.6 0.11 0.10 Hematology assays Blood obtained from all mice groups were analyzed for RBC, WBC, platelets, neutrophils, lymphocytes, and monocyte counts, as well as hemoglobin, HCT and MCV levels.
The results are presented in the Table below. Treatment with the ISIS oligonucleotides did not cause any changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides.
Table 34 Blood cell count in CD1 mice at Day 45 RBC Platelets WBC Neutrophils Lymphocytes Monocytes (x 106/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) PBS 9.6 853 3.9 12 3174 129 863568 10.3 859 7.8 15 6060 347 863581 9.4 812 8.0 17 6162 359 863582 9.1 868 10.5 21 6671 713 863587 9.6 903 9.8 10 8233 344 Table 35 Hematology markers in CD1 mice at Day 45 Hemoglobin HCT MCV
(g/dL) (0/0) (th) PBS 14.5 44 46 863568 16.2 47 46 863581 14.5 43 46 863582 13.5 41 45 863587 15.0 45 47 Body and organ weight measurements To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured every week and are presented in the Table below.
Organ weights were measured and the data is also presented in the Table below.
The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.
Table 36 Body weights (g) in CD1 mice Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 (day 1) (day 9) (day 16) (day 23) (day 30) (day 37) (day 43) Table 37 Organ weights (g) in CD1 mice Liver Spleen Kidney PBS 2.06 0.11 0.59 863568 2.46 0.10 0.54 863581 2.23 0.13 0.56 863582 2.52 0.16 0.63 863587 2.94 0.16 0.64 Study 3 Treatment Groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with 5 mg/kg or 15 mg/kg of ISIS oligonucleotides. One group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
The ISIS oligonucleotides tested are presented in the Table below.
'Unconjugated parent ISIS No.' refers to the ISIS oligonucleotide described in the in vitro studies above with the same sequence. '3'-THA
counterpart ISIS No.' refers to the 3'THA conjugated oligonucleotide with the same sequence and described in the transgenic mice study above.

Table 38 ISIS oligonucleotides tested in CD1 mice tolerability study 3'-THA
Unconjugated SEQ
ISIS No. counterpart Sequence parent ISIS No. ID NO
ISIS No Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).
The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.
Table 39 Plasma chemistry markers in CD1 mice plasma at Day 45 Dose ALT AST ALB BUN CRE TBIL
(mg/kg) (IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 26 62 2.5 19.5 0.08 0.18 5 96 64 2.4 19.3 0.08 0.15 187 115 2.3 22.0 0.07 0.17 5 176 160 2.5 17.2 0.09 0.13 15 621 492 2.5 15.6 0.10 0.20 5 50 50 2.5 19.9 0.10 0.13 15 87 70 2.4 20.3 0.11 0.13 5 321 301 1.7 19.3 0.05 0.19 15 508 472 2.4 20.5 0.09 0.22 5 306 156 2.3 20.0 0.06 0.13 15 373 212 2.4 21.0 0.06 0.14 Hematology assays 15 Blood obtained from all mice groups were analyzed for RBC, WBC, platelets, neutrophils, lymphocytes, and monocyte counts, as well as hemoglobin, HCT and MCV levels.
The results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense oligonucleotides were excluded in further studies.

Table 40 Blood cell count in CD1 mice at Day 45 RBC Platelets WBC Neutrophils Lymphocytes Monocytes Dose (x (x (x (x 103/ L) (x 103/ L) (x 103/ L) (mg/kg) 106/ L) 103/ L) 103/ L) PBS 9.4 1218 8.8 13 7078 845219 5 9.4 1349 7.2 13 5749 15 9.3 1263 7.2 13 5664 863577 5 9.2 1401 8.0 17 5851 15 8.2 1299 10.0 10 7718 863579 5 10.0 1252 9.0 16 6876 15 9.6 1256 8.2 11 6645 863637 5 9.0 956 5.7 14 4266 15 9.3 843 6.8 13 5044 863682 5 9.4 1040 5.9 13 4581 15 9.0 800 8.3 8 6757 Table 41 Hematology markers in CD1 mice at Day 45 Hemoglobin HCT MCV
Dose (mg/kg) (g/dL) ( /0) (th) PBS 14.1 45 48 5 14.2 45 49 13.8 43 46 5 13.9 45 49 15 12.2 39 48 5 14.9 47 48 15 15.3 48 50 5 13.5 42 47 15 13.8 43 47 5 14.5 45 48 15 13.6 43 48 Body and organ weight measurements To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured every week and are presented in the Table below.

Organ weights were measured and the data is also presented in the Table below.
The results indicate that effect of treatment with the antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.

Table 42 Body weights (g) in CD1 mice Dose Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 (mg/kg) (day 1) (day 8) (day 15) (day 22) (day 29) (day 36) (day 42) Table 43 5 Organ weights (g) in CD1 mice Dose (mg/kg) Liver Spleen Kidney PBS 1.84 0.08 0.50 5 2.42 0.13 0.54 15 2.41 0.15 0.54 5 2.47 0.11 0.58 15 2.62 0.15 0.59 5 2.53 0.13 0.57 15 2.82 0.12 0.54 5 2.38 0.17 0.52 15 2.67 0.22 0.58 5 1.79 0.11 0.54 15 2.13 0.21 0.53 Example 6: Tolerability of ISIS oligonucleotides targeting human PCSK9 in Sprague-Dawley rats Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and 10 evaluated for changes in the levels of various plasma chemistry markers.
Treatment Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 15 mg/kg of ISIS oligonucleotide. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Liver function To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).
Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin (TBIL), BUN, albumin (ALB), and creatinine (CRE) were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. Plasma levels of albumin were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in g/dL.
Table 44 Liver function markers in Sprague-Dawley rats ALT AST ALB BUN CRE TBIL
(IU/L) (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 20 64 3.1 13 0.2 0.1 863568 40 70 3.5 14 0.2 0.2 863576 39 94 3.7 16 0.3 0.1 863579 33 91 3.3 14 0.2 0.1 863581 36 154 2.8 18 0.3 0.2 863582 38 122 2.9 20 0.3 0.1 863587 38 87 3.0 12 0.2 0.1 863633 26 78 3.0 16 0.3 0.1 863655 27 84 3.1 15 0.2 0.1 863670 30 91 3.2 15 0.2 0.1 863681 29 81 3.2 14 0.2 0.1 Kidney function To evaluate the effect of ISIS oligonucleotides on kidney function, urine levels of blood creatinine and total protein were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Results are presented in the Table below, expressed in mg/dL.
ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies. 'N/A' indicates that data is not available for that group.

Table 45 Kidney function markers (mg/dL) in Sprague-Dawley rats CRE TP
(mg/dL) (mg/dL) Body and organ weights To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights were measured every week and are presented in the Table below.
Organ weights were measured and the data is also presented in the Table below.
The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.
Table 46 Body weights (g) Baseline Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 (day 1) (day 8) (day 15) (day 22) (day 29) (day 36) (day 43) Table 47 Organ weights (g) Liver Spleen Kidney PBS 12 0.8 3.4 863568 16 1.2 3.3 863576 12 1.3 3.0 863579 11 1.0 3.0 863581 13 2.0 3.1 863582 14 2.1 3.5 863587 14 1.1 3.4 863633 12 1.6 3.2 863655 11 1.7 3.4 863670 12 1.0 2.9 863681 12 1.1 3.3 Example 7: Effect of antisense inhibition of PCSK9 in transgenic mouse model A transgenic mouse model developed at UCI and has never been described in the literature. The human PCSK9 genomic construct contained in fosmid ABC7-611722G24 was restricted with Nhel to produce a DNA fragment containing the entire genomic sequence as well as 8 Kb of 5' and 0.4 Kb of 3' noncoding sequence. PCSK9 transgenic mice were produced by random insertion via micronucleus injection. Progeny expressed human PCSK9 mRNA in the liver and secreted human PCSK9 plasma protein.
Treatment Transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (AS0s) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9%
PBS for injection.
The Tg mice were divided into 27 groups of 3 mice each. Groups received subcutaneous injections of ISIS oligonucleotide at a dose of 0.25 mg/kg, 1.00 mg/kg, or 5.00 mg/kg once per week for 4 weeks. One group of 4 mice received subcutaneous injections of PBS once per week for 4 weeks. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.
RNA analysis On day 26, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA
expression of PCSK9. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN . As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of PCSK9 mRNA in comparison to the PBS control.

Table 48 Percent inhibition of PCSK9 mRNA in the transgenic mice liver relative to the PBS control Weekly %
Dose ED5o Inhibition (mg/kg) 0.25 26 863568 1 76 0.5 0.25 9 863579 1 41 1.4 0.25 12 863581 1 73 0.6 0.25 32 863582 1 71 0.5 0.25 27 863587 1 71 0.5 0.25 60 863633 1 96 0.2 0.25 37 863655 1 94 0.3 0.25 0 863670 1 62 1.0 0.25 25 863681 1 45 0.9 Protein analysis PCSK9 plasma protein was measured by a human-specific ELISA kit (R&D Systems).
Results are 5 presented as percent change of protein levels, relative to PBS control.
As shown in the Table below, treatment with ISIS antisense oligonucleotides resulted in significant reduction of PCSK9 plasma protein levels in comparison to the PBS control.

Table 49 Percent inhibition of PCSK9 plasma protein in the transgenic mice relative to the PBS control Weekly %
Dose ED5o Inhibition (mg/kg) 0.25 37 863568 1 74 0.4 0.25 5 863579 1 41 1.5 0.25 31 863581 1 59 0.6 0.25 25 863582 1 73 0.5 0.25 33 863587 1 77 0.4 0.25 50 863633 1 97 0.3 0.25 44 863655 1 93 0.3 0.25 23 863670 1 38 1.6 0.25 28 863681 1 54 0.8 LDL-cholesterol levels Levels of LDL-cholesterol in the plasma were measured by an automated clinical chemistry analyzer 5 (Hitachi Olympus AU400e, Melville, NY). Levels of LDL receptor (LDLr) protein in the liver were also measured. Results are presented as percent change of levels, relative to PBS
control. As shown in the Table below, treatment with several ISIS antisense oligonucleotides resulted in significant reduction of levels of LDL-cholesterol in the plasma in comparison to the PBS control.
Correspondingly, it was observed that treatment with ISIS antisense oligonucleotides resulted in dose-dependent liver LDLr protein increases in the mice.

Table 50 Inhibition of LDL-cholesterol levels in the plasma relative to the PBS control Weekly %
Dose inhibition (mg/kg) 0.25 7 0.25 0 0.25 30 0.25 23 0.25 20 0.25 27 0.25 29 0.25 19 0.25 21 Table 51 5 Percent increase of liver LDLr in the transgenic mice relative to the PBS control Weekly %
Dose increase (mg/kg) 0.25 3 0.25 -46 0.25 -44 0.25 -19 0.25 -6 0.25 -22 0.25 0 0.25 9 0.25 123 Example 8: Measurement of viscosity of antisense oligonucleotides targeting human PCSK9 The viscosity of select antisense oligonucleotides from the studies described above was measured with the aim of screening out antisense oligonucleotides which have a viscosity of more than 40 centipoise 5 (cP). Oligonucleotides having a viscosity greater than 40 cP would have less than optimal viscosity.
Oligonucleotides (32-35 mg) were weighed into a glass vial, 120 tiL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50 C. Part (75 tit) of the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25 C and the viscosity of the sample was measured. Another part (20 viL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85 C (Cary UV
instrument). The results are presented in the Table below, where the concentration of each antisense oligonucleotide was 350 mg/ml, and indicate that all the antisense oligonucleotides solutions were optimal in their viscosity under the criterion stated above.

Table 52 Viscosity of antisense oligonucleotides targeting human PCSK9 Viscosity Concentration ISIS No (cP) (mg/mL) Example 9: Effect of ISIS antisense oligonucleotides targeting human PCSK9 in cynomolgus monkeys Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide efficacy and tolerability, as well as their pharmacokinetic profile in the liver and kidney, were evaluated.
At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database;
therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (the complement of GENBANK
Accession No.
NW_005092960.1 truncated from nucleotides 83474000 to 83501000, designated herein as SEQ ID NO:
1544). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide to SEQ ID NO: 1544 is presented in the Table below. "Start site" indicates the 5'-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence.
Table 53 Antisense oligonucleotides complementary to the rhesus PCSK9 genomic sequence (SEQ ID NO: 1544) Target Target SEQ ID
ISIS No Start Mismatches Stop NO
Site Site Treatment Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed 2-4 kg.
Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS
oligonucleotide or PBS in clockwise rotation between four different sites on the backs of the monkeys (i.e. left, right, top, and bottom) on each day of dosing; one site per dose. The monkeys were dosed twice a week (days 1, 5, 9, and 14) for the first two weeks, and then subsequently once a week for 10 weeks (days 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84) with 10 mg/kg of ISIS oligonucleotide. A control group of 5 cynomolgus monkeys was injected with PBS
in a similar manner and served as the control group.
During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. Scheduled euthanasia of the animals was conducted on day 86 approximately 48 hours post-dose by exsanguination while under deep anesthesia. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).
Target Reduction RNA analysis On day 86, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA
expression of PCSK9. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN . As shown in the Table below, treatment with ISIS antisense oligonucleotides, ISIS
863633, ISIS 863670, and ISIS 863681, which had no mismatches with the rhesus gene, resulted in significant reduction of PCSK9 mRNA in comparison to the PBS control.

Table 54 Percent inhibition of PCSK9 mRNA in the cynomolgus monkey liver relative to the PBS control %
inhibition Protein analysis Approximately 1 mL of blood was collected from all available animals on day -7 (pre-treatment), Days 16, 30, 58, and 86 (approximately 48 hours post-dose on Days 14, 28, 56, and 84, respectively) and placed in tubes containing the potassium salt of EDTA. The tubes were centrifuged (3000 rpm for 10 min at 4 C) to obtain plasma. The results are presented in the Table below as a percentage increase or decrease over the levels at Day -7 (pre-treatment levels). As shown in the Table below, treatment with ISIS antisense oligonucleotides, ISIS 863633, ISIS 863670, and ISIS 863681, which had no mismatches with the rhesus gene, resulted in significant reduction of PCSK9 protein levels.
Liver tissue was also analyzed for levels of LDL receptor (LDLr) protein induction levels. The results are presented in the Table below. The data demonstrates that treatment with the three antisense oligonucleotides that are homologous to the rhesus monkey genomic sequence resulted in hepatic LDLr protein induction after 12 weeks of treatment.
Table 55 Plasma protein levels ( /0 of pre-treatment levels) in the cynomolgus monkey Day 16 Day 30 Day 86 Table 56 Hepatic LDLr protein levels ( /0 of PBS control) in the cynomolgus monkey `)/0 increase Plasma lipid levels To evaluate the effect of ISIS oligonucleotides on total cholesterol, LDL-Cholesterol, HDL-cholesterol, and triglyceride levels, monkeys were fasted overnight prior to blood collection. Approximately 1.7 mL of blood samples were collected from all the study groups. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3,000 rpm for 10 minutes at room temperature to obtain serum. Levels were measured using a Toshiba 200FR NE0 chemistry analyzer (Toshiba Co., Japan). The data demonstrates that treatment with the three antisense oligonucleotides with homology with the rhesus monkey genomic sequence resulted in significant reduction of plasma total cholesterol and LDL-cholesterol. As expected, reduction of human PCSK9 levels did not affect plasma HDL-cholesterol or triglyceride levels.
Table 57 Plasma lipid levels (mg/dL) in cynomolgus monkey on Day 86 Total LDL- HDL-Triglycerides cholesterol cholesterol cholesterol Tolerability studies Clinical Chemistry Parameters To evaluate the effect of ISIS oligonucleotides on hepatic function, monkeys were fasted overnight prior to blood collection. Approximately 1.7 mL of blood samples were collected from all the study groups.
Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3,000 rpm for 10 minutes at room temperature to obtain serum. Levels of total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and albumin (ALB) were measured using a Toshiba 200FR
NE0 chemistry analyzer (Toshiba Co., Japan). The results indicate that antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides.
Specifically, ISIS 863633 was observed to be well tolerated.
Table 58 Plasma Chemistry markers in cynomolgus monkey on Day 86 ALT AST Bilirubin ALB
(IU/L) (IU/L) (mg/dL) (g/dL) PBS 65 64 0.2 4.1 863655 63 59 0.3 3.5 863633 47 70 0.3 4.0 863568 44 64 0.3 4.1 863579 50 52 0.2 4.1 863582 42 57 0.2 4.2 863581 55 61 0.3 4.2 863587 110 76 0.3 3.9 863670 42 64 0.3 4.1 863681 62 60 0.3 4.1 Kidney function To evaluate the effect of ISIS oligonucleotides on kidney function, monkeys were fasted overnight prior to blood collection. Approximately 1.7 mL of blood samples were collected from all the study groups.
Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3,000 rpm for 10 minutes at room temperature to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NE0 chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL.
The plasma chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides.
Specifically, ISIS 863633 was observed to be well tolerated.

Table 59 Plasma BUN and creatinine levels (mg/dL) in cynomolgus monkeys at day 86 BUN Creatinine PBS 31 0.8 863655 26 0.8 863633 27 0.8 863568 27 0.9 863579 24 0.8 863582 24 0.8 863581 27 0.8 863587 24 0.8 863670 26 1.0 863681 23 0.8 Hematology To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, approximately 0.5 mL of blood was collected from each of the available study animals in tubes containing K2-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA). The data is presented in the Tables below.
The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose. Specifically, ISIS 863633 was observed to be well tolerated.
Table 60 Blood cell counts in cynomolgus monkeys on day 86 RBC Platelets WBC
Neutrophils Lymphocytes Monocytes (x 106/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) (x 103/ L) PBS 5.5 340 9.0 3.1 5.4 0.3 863655 5.6 361 9.7 3.7 5.4 0.3 863633 5.6 395 11.4 4.5 6.5 0.3 863568 5.8 409 12.1 602 5.4 0.3 863579 5.9 390 8.0 2.2 5.3 0.3 863582 5.6 333 8.9 3.0 5.5 0.3 863581 5.8 382 8.2 2.4 5.3 0.3 863587 6.0 348 8.8 2.3 6.0 0.2 863670 5.4 417 10.7 4.3 5.9 0.3 863681 5.6 408 9.0 3.1 5.5 0.2 Table 61 Hematologic parameters in cynomolgus monkeys on day 86 Hemoglobin HCT
(g/dL) (0/0) PBS 12.8 45 863655 13.0 43 863633 12.9 43 863568 13.0 44 863579 13.5 46 863582 12.4 43 863581 13.0 44 863587 13.5 46 863670 12.7 43 863681 12.7 43 Overall, the results of the study indicate that ISIS 863633 is the most potent and well tolerated .. compound of those tested for inhibiting PCSK9 and is an important candidate for the treatment of cardiovascular diseases, specifically for lowering LDL-cholesterol levels.
Example 10: Comparative inhibition of antisense oligonucleotides targeting human PCSK9 in a dose response assay The unconjugated parent oligonucleotides of th antisense oligonucleotides tested in the monkey study were compared with previously designed compounds. ISIS 848542, ISIS 848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040, ISIS 849171, and ISIS 849236 were tested for efficacy with ISIS
405879 and ISIS 405995, which have previously been determined to be some of the most potent antisense compounds in vitro (see e.g., U.S. Patent 8,084,437), as well as ISIS 431131 and ISIS 480604, which have been previously described in U.S. Patent 9,127,276.
Cells were plated at a density of 10,000 cells per well and 39.1 nM, 156.25 nM, 625 nM, 2,500 nM, and 10,000 nM concentrations of antisense oligonucleotide was added to the medium for free uptake of oligonucleotide by the cells. After a treatment period of approximately 48 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer probe set from ABI (ID# Hs03037355_ml) was used to measure mRNA levels. PCSK9 mRNA
levels were adjusted according to total RNA content, as measured by RIBOGREEN . Results are presented as percent inhibition of PCSK9, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented. PCSK9 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. ISIS
848542, ISIS 848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS
849040, ISIS 849171, and ISIS 849236 demonstrated superior efficacy compared to all previously disclosed oligonucleotides.

Table 62 39.1 156.25 625.0 2500.0 10000.0 IC50 ISIS No nM nM nM nM nM ( M) 405879 0 0 0 0 0 >10 405995 0 0 0 0 0 >10 431131 0 3 0 0 5 >10 480604 0 1 19 31 36 >10 848542 9 37 70 83 88 0.4 848593 19 36 51 53 69 1.1 848597 41 67 78 86 91 0.04 848598 41 64 76 85 90 0.1 848630 0 17 59 79 85 0.9 848833 4 66 88 93 94 0.2 849040 41 93 98 99 98 <0.03 849171 0 52 86 94 96 0.3 849236 0 26 71 89 95 0.6

Claims (66)

WHAT IS CLAIMED:
1. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 3-1540.
2. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 3-1540.
3. A compound comprising a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 3-1540.
4. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length complementary within nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 of SEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, 90%, 95%, or 100% complementary to SEQ ID NO: 2
5. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion of nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, 90%, 95%, or 100% complementary to SEQ ID NO: 2.
6. A compound comprising a modified oligonucleotide complementary with an equal portion of nucleobases 6356-6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 of a PCSK9 nucleic acid having the nucleobase sequence of SEQ ID
NO: 2.
7. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 6356-6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 of a PCSK9 nucleic acid having the nucleobase sequence of SEQ ID NO: 2, wherein the nucleobase sequence of the modified oligonucleotide is complementary to SEQ ID NO: 2.
8. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length comprising at least an 8 nucleobases portion of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.
9. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NO: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.
10. A compound comprising a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353.
11. A compound comprising a modified oligonucleotide 8 to 80 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
12. The compound of any one of claims 1-11, wherein the oligonucleotide is at least 80%, 85%, 90%, 95% or 100% complementary to SEQ ID NO: 2.
13. The compound of any one of claims 1-12, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase.
14. The compound of claim 13, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
15. The compound of claim 13 or 14, wherein the modified sugar is a bicyclic sugar.
16. The compound of claim 15, wherein the bicyclic sugar is selected from the group consisting of: 4'-(CH2)-O-2' (LNA); 4'-(CH2)2-O-2' (ENA); and 4'-CH(CH3)-O-2' (cEt).
17. The compound of claim 13 or 14, wherein the modified sugar is 2'-O-methoxyethyl.
18. The compound of any one of claims 13-17, wherein the modified nucleobase is a 5-methylcytosine.
19. The compound of any one of claims 1-18, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides;

wherein the gap segment is positioned immediately adjacent to and between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
20. The compound of any one of claims 1-19, wherein the compound is single-stranded.
21. The compound of any one of claims 1-19, wherein the compound is double-stranded.
22. The compound of any one of claims 1-21, wherein the compound comprises ribonucleotides.
23. The compound of any one of claims 1-21, wherein the compound comprises deoxyribonucleotides.
24. The compound of any one of claims 1-23, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides.
25. The compound of any one of claims 1-23, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.
26. The compound of any one of claims 1-23, wherein the modified oligonucleotide consists of 15 to 30 linked nucleosides.
27. A compound comprising a modified oligonucleotide 16 linked nucleosides in length having a nucleobase sequence consisting of any one of SEQ ID NOs: 1016, 1528, 763, 1071, 1147, 1149, 1016, 955, 1195, and 353, wherein the modified oligonucleotide comprises a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of three linked nucleosides; and a 3' wing segment consisting of three linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment; wherein the 5' wing segment and the 3' wing segment comprise cEt sugars; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.
28. A compound comprising a modified oligonucleotide according to the following formula: Aks Aks Tks Ads Ads Tds mCds Tds mCds Ads Tds Gds Tds mCks Aks Gk; wherein, A = an adenine, mC = a 5-methylcytosine G = a guanine, T = a thymine, k = a cEt modified nucleoside, d = a 2' -deoxynucleoside, and s = a phosphorothioate internucleoside linkage.
29. The compound of any preceding claim, further comprising a conjugated moiety and a conjugate linker.
30. The compound of claim 29, wherein the conjugate group comprises a GalNAc cluster comprising 1-3 GalNAc ligands.
31. The compound of claim 29 or claim 30, wherein the conjugate linker consists of a single bond.
32. The compound of claim 31, wherein the conjugate linker is cleavable.
33. The compound of claim 32, wherein the conjugate linker comprises 1-3 linker-nucleosides.
34. The compound of any one of claims 29-33, wherein the wherein the conjugate group is attached to the modified oligonucleotide at the 5' -end of the modified oligonucleotide.
35. The compound of any one of claims 29-33, wherein the wherein the conjugate group is attached to the modified oligonucleotide at the 3' -end of the modified oligonucleotide.
36. A compound according to the following formula :

37. A compound consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO: 1016, wherein the modified oligonucleotide consists of a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of three linked nucleosides; and a 3' wing segment consisting of three linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; wherein each cytosine is a 5-methylcytosine; and wherein the conjugate group is 5'-Trishexylamino-(THA)-C6GalNAC3 and is positioned at the 5'end of the modified oligonucleotide.
38.
A compound consisting of a pharmaceutically acceptable salt of any of the compounds of claims 1-37.
39. The compound of claim 38, wherein the pharmaceutically acceptable salt is a sodium salt.
40. The compound of claim 38, wherein the pharmaceutically acceptable salt is a potassium salt.
41. A composition comprising the compound of any one of claims 1-40 and a pharmaceutically acceptable carrier.
42. A composition comprising a compound or modified oligonucleotide of any preceding claim, for use in therapy.
43. A method of treating, preventing, or ameliorating a disease associated with PCSK9 in an individual comprising administering to the individual a compound targeted to PCSK9, thereby treating, preventing, or ameliorating the disease.
44. The method of claim 43, wherein the compound is an antisense compound targeted to PCSK9.
45. The method of claim 43 or 44, wherein the disease is hypercholesterolemia or dyslipidemia.
46. The method of any of claims 43-45, wherein administering the compound inhibits or reduces LDL-cholesterol levels and total cholesterol levels, and improves the induction of hepatic LDL receptor levels.
47. A method of inhibiting expression of PCSK9 in a cell comprising contacting the cell with a compound targeted to PCSK9, thereby inhibiting expression of PCSK9 in the cell.
48. The method of claim 47, wherein the cell is in the liver of an individual.
49. The method of claim 48, wherein the individual has, or is at risk of having, hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
50. A method of reducing or inhibiting LDL-cholesterol levels and total cholesterol levels in an individual having, or at risk of having, a disease associated with PCSK9 comprising administering a compound targeted to PCSK9 to the individual, thereby reducing or inhibiting LDL-cholesterol levels and total cholesterol levels in the individual.
51. The method of claim 50, wherein the individual has, or is at risk of having, hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
52. The method of any one of claims 43-51, wherein the compound is an antisense compound targeted to PCSK9.
53. The method of any one of claims 43-52, wherein the compound is the compound of any one of claims 1-40 or composition of any of claims 41 and 42.
54. The method of claim 52 or 53, wherein the compound is administered parenterally.
55. Use of a compound targeted to PCSK9 for treating, preventing, or ameliorating a disease associated with PCSK9.
56. The use of claim 55, wherein the disease is hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
57. The use of claim 55 or 56, wherein the compound is an antisense compound targeted to PCSK9.
58. The use of any one of claims 55-57, wherein the compound is the compound of any one of claims 1-40 or composition of any of claims 41 and 42.
59. Use of a compound targeted to PCSK9 in the manufacture of a medicament for treating, preventing, or ameliorating a disease associated with PCSK9.
60. The use of claim 59 wherein the disease is hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
61. The use of claim 59 or 60, wherein the compound is an antisense compound targeted to PCSK9.
62. The use of any one of claims 59-61, wherein the compound is the compound of any one of claims 1-40 or composition of any of claims 41 and 42.
63. Use of a compound targeted to PCSK9 in the preparation of a medicament for treating, preventing, or ameliorating a disease associated with PCSK9.
64. The use of claim 63, wherein the disease is hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.
65. The use of claim 63 or 64, wherein the compound is an antisense compound targeted to PCSK9.
66. The use of any one of claims 63-65, wherein the compound is the compound of any one of claims 1-40 or composition of any of claims 41 and 42.
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